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LABORATORY  WORK 


IN 


PHYSIOLOGICAL  CHEMISTRY. 


BY 
FREDERICK   G.   NOW,   Sc.D.,   M.D., 

JUNIOR    PROFESSOR    OF     HYGIENE   AND   PHYSIOLOGICAL   CHEMISTRY, 
UNIVERSITY  OF  MICHIGAN. 


SECOND    EDITION    REVISED   AND    ENLARGED. 
WITH  FRONTISPIECE  AND   TWENTY-FOUR    ILLUSTRATIONS. 


ANN  ARBOR  : 

George  Wahr,  Publisher, 

i8q8. 


Copyright,  1898,  by  F.  G.  Novy. 


QP519 


The  Inland  Press,  Ann  Arbor,  Mich, 


PREFACE. 

This  edition  is  greatly  enlarged  and  indeed  wholly 
re-written.  The  directions  for  laboratory  work  cover  the 
three  great  food-stuffs,  and  the  fluids  and  secretions  of  the 
body.  Brief  explanatory  descriptions  of  the  various  sub- 
stances and  secretions  studied  are  added  in  order  to  give 
the  student  a  better  survey  of  the  subject  matter  as  a  whole. 

Every  medical  student  should  receive  thorough  drill  in 
the  laboratory,  not  merely  in  so-called  urine  analysis,  but  in 
the  broader  field  of  physiological  chemistry.  He  should  be 
taught  to  observe  and  to  reason;  to  correlate  the  facts 
brought  out  in  the  laboratory  in  their  relation  to  physiology, 
hygiene  and  disease.  These  notes  have  been  prepared  with 
the  object  of  furthering  such  study,  and  it  is  hoped  that 
they  will  prove  as  useful  to  others  as  to  the  author's  own 
classes  in  the  University  of  Michigan.  The  experimental 
work  laid  down  in  the  following  pages  has  been  repeatedly 
verified  in  actual  student  work  and  the  directions  given  are 
such  as  to  insure  results.  The  author's  classes  devote  the 
entire  afternoon,  daily,  for  three  months  to  this  work. 
Even  with  this  amount  of  time  at  their  disposal  certain 
parts  of   physiological  chemistry  must  be  left  untouched. 

The  works  of  SalkowsM,  Hammarsten,  v.  Noorden  and 
others  have  been  freely  drawn  upon  for  suggestions  in  the 


4  PREFACE. 

preparation  of  these  directions.  Thanks  are  due  to  the 
Bausch  and  Lomb  Optical  Co.,  of  Rochester,  for  kindly- 
supplying'  several  cuts.  I  wish  also  to  acknowledge  the 
invaluable  help  rendered  me  by  way  of  suggestions, 
verification  of  experimental  details,  and  proof-reading  by 

my  able  assistant,  Mr.  C.  L.  Bliss. 

F.  G.  NOVY. 

Ann  Arbor,  Mich.,  August  1,  1898. 


Fats 


CONTENTS. 


CHAPTER   I. 


CHAPTER   II. 


Carbohydrates 


15 


CHAPTER   III. 


Proteins 


37 


CHAPTER   IV. 


Saliva 


.    55 


CHAPTER   V, 


Gastric  Juice    . 


,    61 


CHAPTER  VI. 


Pancreatic  Secretion 


76 


CHAPTER   VII. 


Bile 


87 


CHAPTER   VIII, 


Blood 


97 


CHAPTER  IX. 


Milk   , 


116 


CHAPTER   X. 


riu.NT. 


<  I  !  I  I 


123 


6  CONTENTS. 

CHAPTER   XI. 

Quantitative  Analysis— 

Urine,  Milk,  Gastric  Juice,  Blood        .         .         .         .         .         .217 

CHAPTER  XII. 

Tables  for  Examination  op  Urine 283 

List  of  Reagents 317 

Index 321 


CHAPTER    I. 
FATS. 

Fats  are  widely  distributed  in  the  animal  and  in  the 
vegetable  kingdom.  Indeed  it  may  be  said  that  proto- 
plasm always  contains  some  fat,  and  every  cell,  therefore, 
has  more  or  less  of  these  compounds.  In  animals  the  fat 
is  usually  stored  up  in  the  subcutaneous  tissue,  or  about 
the  abdominal  organs.  In  plants  the  seed,  or  fruit,  or  even 
the  roots  are  rich  in  fats.  Many  bacteria  contain  an  appre- 
ciable amount  of  fat  stored  up  within  their  walls.  This  is 
especially  true  of  the  tubercle  bacillus,  which  in  the  dry 
state  may  contain  as  much  as  40  per  cent,  of  fat.  Its 
characteristic  behavior  to  stains  is  probably  due  to  this 
large  amount  of  fat. 

Fats  are  neutral  compounds,  or  esters,  resulting  from 
the  union  of  glycerin,  CH2OH .  CHOH .  CH2OH,  a  triatomic 
alcohol,  with  a  mono-basic  fatty  acid.  The  hydrogen 
of  each  of  the  hydroxyl  groups  in  glycerin  is  replaced 
by  a  fatty  acid  radical.  If  the  fatty  acid  is  stearic  acid 
the  resulting  fat  is  tri-stearin,  or  stearin.  Similarly  with 
palmitic  and  oleic  acids,  we  have  the  corresponding  fats 
palmitin  and  olein. 

Ordinary  fats  are  really  mixtures  of  several  fats.  Thus 
the  animal  fats  are  mixtures  of  variable  amounts  of  stearin, 
palmitin  and  olein.  Stearin  has  the  higher  melting  point 
and  when  present  in  appreciable  quantity  it  makes  a  rela- 
tively hard  fat  as  tallow,  or  suet.  On  the  other  hand,  olein 
is  a  liquid  fat  at  ordinary  temperature  and  mixtures  of 
this  with  palmitin  yield  a  soft  fat  as  lard.  Olive  oil  and 
other  vegetable  oils  can  be  considered  as  nearly  pure  olein, 

although  glycerides  of  various  other  fatty  acids  may  be 

•i 


8  PHYSIOLOGICAL  CHEMISTRY. 

present.  Milk-fat  or  butter  consists  of  the  three  glycer- 
ides  mentioned  above  and  small  quantities  of  glycerides  of 
butyric  and  other  fatty  acids.  The  butyric  acid  and  lower 
fatty  acids  are  soluble  in  water  and  volatile,  and  consti- 
tute about  six  per  cent,  of  the  butter  fat.  Inasmuch  as 
lard  and  tallow  practically  have  no  volatile  fatty  acids, 
this  fact  is  utilized  in  the  analysis  of  butter  for  adultera- 
tion. 

Fats  when  pure  are  colorless,  or  nearly  so;  tasteless 
and  odorless,  and  are  insoluble  in  and  lighter  than  water. 
They  are  soluble  in  boiling-  absolute  alcohol  but  recrystal- 
lize  on  cooling'.  They  are  readily  soluble  in  ether,  chloro- 
form, and  benzol.  They  are  emulsified  or  brought  into  a 
fine  state  of  division  by  soap,  gum-arabic,  and  by  albumin- 
ous bodies. 

Furthermore,  fats  when  pure  are  neutral  in  reaction. 
As  the  result  of  exposure  to  physical,  chemical  and  living 
agents  they  are  readily  split  into  their  component  parts, 
that  is  glycerin  and  fatty  acids.  The  change  that  takes 
place  is  one  of  hydration,  and  whenever  an  ester  of  any 
kind  is  thus  decomposed  it  is  said  to  undergo  saponifica- 
tion. This  change  for  stearin  is  indicated  by  the  following 
equation: — 

ch2o.co.c17h35  ch2oh 

^ho  .co.c17h35  +  3  hoh  =  choh  +  3  c17h35co.oh. 

6h,o.co.c17h35  6h2oh 

This  hydration  of  a  neutral  fat  can  be  brought  about 
by  steam  under  pressure;  by  boiling  with  water  containing 
a  small  amount  of  an  acid,  as  sulphuric  acid  (manufacture 
of  glycerin,  and  of  stearic  acid  or  "commercial  stearin" 
used  in  making  of  candles);  by  boiling  with  an  alkali,  as 
potash,  soda,  lime  or  lead  oxide  (manufacture  of  soap,  lead 
plaster);  by  the  action  of  air  and  light  (rancidity  of  fats); 
by  the  action  of  bacteria;  and  by  the  action  of  enzymes,  as 
that  contained  in  the  pancreatic  secretion. 


FATS.  9 

The  fat  taken  in  as  food  is  not  absorbed  until  it  reaches 
the  intestines,  where  it  is  acted  upon  by  the^pancreatic 
juice.  Owing-  to  the  action  of  the  fat-splitting-  ferment 
contained  in  this  secretion,  a  portion  of  the  fat  undergoes 
cleavage  into  glycerin  and  fatty  acid.  The  latter  com- 
bines with  the  sodium  carbonate  present  to  form  a  sodium 
soap.  A  small  amount  of  this  soap  emulsifies  a  large 
amount  of  fat,  the  fat  is  thus  divided  into  very  minute 
globules  and  in  this  form  it  becomes  absorbed.  Many 
bacteria  exercise  a  similar  fat-splitting  action  and  hence 
fatty  acids  may  be  formed  in  the  intestines  by  their  activ- 
ity. The  bile  secretion  assists  in  the  absorption  of  fat, 
and  when  bile  cannot  pass  into  the  intestines  a  large 
amount  of  fat,  chiefly  as  fatty  acids,  is  excreted  with  the 
feces.  The  cells  in  the  intestinal  wall  seem  to  have  power 
of  synthesizing  fats,  out  of  glycerin  and  fatty  acids.  This 
action  is  analogous  to  the  synthesis  by  these  same  cells  of 
pepton  to  albumin. 

The  fat  deposited  in  the  body  is  derived  in  part  directly 
from  the  fat  present  in  the  food  and  absorbed  as  such.  In 
addition  to  this  some  fat  is  made  in  the  body  out  of  the 
protein  and  carbohydrate  molecules.  The  formation  of  fat 
out  of  albuminous  material  is  seen  in  the  fatty  degenera- 
tion of  various  organs  and  tissues  in  disease.  Moreover 
fatty  acids  can  be  obtained  from  albuminous  substances  by 
cleavage  in  the  laboratory,  and  by  bacterial  decomposition. 
The  mass  of  fat,  or  rather  of  fatty  acids  and  soaps,  known 
as  adipocire  and  found  in  decomposing  cadavers,  probably 
owes  its  origin  to  the  latter  form  of  decomposition. 

The  fat  deposited  in  the  tissues  serves  as  a  reserve 
food  to  generate  heat  and  energy.  The  large  amount  of 
carbon  and  hydrogen  contained  in  the  fat  molecule  explains 
the  large  amount  of  heat  generated  when  fat  is  oxidized 
and  the  need  of  much  fat  as  food  in  a  cold  climate.  Fat, 
moreover,  is  a  non-conductor  of  heat  and  thus  serves  to 
prevent  undue  loss  of  heat  from  the  body.  In  starvation 
fat  disappears  rapidly  from  the  tissues. 


10 


PHYSIOLOGICAL   CHEMISTRY. 


The  chyle  and  lymph  may  contain  from  0.5  to  5  per 
cent,  of  fat.  In  certain  diseases,  as  in  fevers,  tubercu- 
losis, etc.,  the  amount  of  fat  present  in  the  blood  is 
increased.  The  blood,  lymph  and  transsudates  contain  an 
appreciable  quantity  of  fatty  acids  as  a  soap.  Stearic  and 
palmitic  acids  are  met  with  as  needle-shaped  crystals  in 
decomposing-  pus,  in  sputum  from  gangrene  of  the  lung, 
and  from  tuberculosis.  For  the  occurrence  of  fats  and 
fatty  acids  in  urine,  see  that  chapter. 

A  mixture  of  stearic  and  palmitic  acids  is  commonly 
called  margaric  acid,  and  a  similar  mixture  of  stearin  and 
palmitin  is  designated  as  margarine.  Oleo-margarine,  a 
substitute  for  butter,  is  essentially  purified  animal  fat. 

1. — Preparation  of  Pure  Fat. — For  this  experiment  the  students 
will  use,  alternately,  pork-fat  and  suet.  Cut  up  10  g.  of  subcut- 
aneous pork  fat,  or  of  suet,  into  as  small  pieces  as  possible.  Place 
into  a  small  evaporating'  dish,  2%. — 3  inches  in  diam- 
eter, and  cautiously  heat  over  a  small  flame,  stirring" 
continually  with  a  thermometer.  Keep  the  tem- 
perature at  120° — 130°  *  for  about  10  minutes.  Then 
strain  through  a  small  piece  of  muslin  and  squeeze 
thoroughly,  receiving  the  clear  fat  into  an  evapor- 
ating dish  (4  inch).  Transfer  the  residue  to  a  small 
mortar,  add  about  5  c.c.  of  strong  alcohol  and  rub 
up  to  a  fine  powder.  Transfer  the  suspension  to  a 
75  c.c.  Erlenmeyer  flask,  and  rinse  out  the  mortar 
with  several  successive  small  portions  of  alcohol. 
Insert  into  the  neck  of  the  flask  a  stopper  provided 
with  a  glass  tube  (Fig.  1)  about  24  inches  long  (con- 
densing tube).  Heat  on  the  water-bath  to  boiling  for 
10  minutes.  Then  set  aside  and,  when  the  suspended 
particles  have  settled,  decant  the  clear  alcohol  onto 
a  small  filter  and  receive  the  alcoholic  filtrate  in  the 
evaporating  dish  containing  the  bulk  of  the  fat. 
To  the  insoluble  residue  remaining  in  the  flask,  add 
Fig,  l.  20  c.c.  of   ether;   insert  the   condensing  tube,   and 

cautiously  boil  on  the  water  bath  for  about  5  minutes.     Then  transfer 
the  entire  contents  of  the  flask  to  the  filter  previously  used  and  receive 

*  The  temperature  figures  given  in  this  work  are  Centigrade.  Unless  otherwise 
indicated,  distilled  water  is  always  to  be  used  for  dilution  and  solution.  The  reactions  are 
to  be  carried  out  in  a  test-tube  unless  specified  otherwise. 


FATS.  11 

the  ethereal  filtrate  in  the  evaporating  dish.  Finally  wash  the  resi- 
due with  a  little  ether,  close  the  filter  and  squeeze  out  most  of  the 
ether.  Then  spread  the  filter  and  allow  the  remaining  ether  to  evap- 
orate spontaneously.  Save  this  yellowish  residue  of  connective  tissue 
for  subsequent  experiments.     (Chapter  III,  1,  2,  3). 

The  evaporating  dish  now  contains  the  strained  fat,  also  the 
alcoholic  and  ethereal  filtrates.  Place  it  on  a  water-bath  and  heat 
carefully  till  all  the  alcohol,  ether  and  water  have  been  driven  off  and 
only  the  pure  fat  remains. 

2. — Place  a  little  of  the  pure  fat,  obtained  as  above,  on  a  slide, 
cover  and  examine  under  the  microscope.  Observe  that  the  little 
round  bodies  are  composed  of  minute  crystals.  These  are  more  dis- 
tinct in  beef  fat. 

3. — Transfer  a  piece  of  fat,  size  of  a  pea,  by  means  of  a  glass 
rod  to  a  test-tube.  Add  5  c.c.  of  a  mixture  of  equal  parts  of  alcohol 
and  ether  and  warm  gently  till  dissolved.  Then  set  aside  for  an  hour 
or  more  and  when  a  deposit  forms  transfer  some  of  it  by  means  of  a 
pipette  to  a  glass  slide,  cover  and  examine  under  the  microscope. 
Sketch  the  crystals  obtained  thus  from  tallow  and  from  lard.  Which 
of  these  two  fats  crystallizes  more  rapidly  ?     Why  ? 

•4. — Transfer  a  piece  of  fat  to  a  test-tube,  add  5  c.c.  of  alcohol 
and  heat  till  dissolved.  Then  introduce  a  strip  of  blue  litmus  paper, 
or  add  a  drop  of  an  aqueous  solution  of  litmus.  Better  still,  to  some 
of  the  alcoholic  solution  add  a  drop  of  alcoholic  rosolic  acid:  a  yel- 
lowish color  indicates  an  acid  reaction.  What  is  the  reaction  of  nor- 
mal fat  ?     Why  do  fats  become  rancid  on  standing  for  some  time  ? 

5. — Place  a  small  piece  of  fat  on  a  filter  paper  and  warm  gently 
over  a  flame,  or  on  a  heated  plate  till  the  fat  melts  and  is  absorbed. 
Note  the  transparent  condition  of  the  paper.  Other  substances  such 
as  glycerin,  paraffin,  which  fill  the  pores  of  the  paper  have  a  similar 
action. 

6. — Rub  up  thoroughly  in  a  mortar  a  piece  of  fat  with  some 
KHS04.  Transfer  the  mixture  to  a  dry  test-tube  and  heat  cautiously. 
The  peculiar  irritating  odor  or  sensation  is  due  to  acrolein  or  acrylic 
aldehyde,  which  is  formed  by  dehydration  from  the  glycerin  of  the 

fat. 

Glycerin,  CH,OH .  CHOH .  CH2OH. 
Acrolein,  CH2  :  CH .  COH. 

7. — To  a  small  piece  of  fat  in  a  test-tube  add  about  10  c.c.  of  a 
semi-saturated  solution  of  sodium  carbonate,  warm  and  shake  thor- 
oughly.    The  liquid  becomes  milky,  but  on  standing,  most  of  the  fat 


12  PHYSIOLOGICAL,  CHEMISTRY. 

collects  on  the  surface.     The  liquid  below  shows  but  slight  cloudiness. 
Neither  emulsion,  solution,  or  saponification  has  taken  place. 

8. — Saponification. — Melt  the  fat  that  is  left  from  the  preced- 
ing- experiments  and  transfer  it  to  a  150  c.c.  Erlenmeyer  flask.  Then 
add  20 — 30  c.c.  of  alcohol  and  3  g.  of  KOH.  Insert  a  condensing  tube 
and  heat  on  the  water-bath  for  about  a  half  an  hour.  Saponification 
takes  place  rapidly.  To  ascertain  if  the  change  is  complete  pour  a 
little  of  the  alcoholic  fluid  into  a  few  c.c.  of  water.  The  liquid  must 
remain  clear.  If  it  becomes  cloudy  it  is  due  to  oil  drops  and  shows 
that  the  saponification  is  incomplete.  The  solution  eventually  con- 
tains soap,  glycerin,  and  excess  of  alkali  and  alcohol. 

9.— Separation  of  the  Fatty  Acids. — To  about  100  c.c.  of  water  in 
a  small  beaker  add  3  c.c.  of  H2SCv  Then  warm  to  about  50°.  Pour 
the  soap  solution,  gradually,  and  with  constant  stirring,  into  the 
warm  acid  liquid.  The  fatty  acids  are  set  free  and  rise  to  the  sur- 
face, forming  a  clear,  oily  liquid.  Place  the  beaker  on  a  water-bath 
and  heat  till  the  aqueous  liquid  below  the  fatty  acid  layer  becomes 
almost  clear,  and  all  the  fatty  acid  has  risen  to  the  surface.  At  the 
same  time  prepare  about  400  c.c.  of  boiling  water. 

Then  transfer  the  contents  of  the  beaker  to  a  small  filter,  previ- 
ously moistened  with  hot  water.  The  fatty  acids,  while  still  liquid, 
are  washed  on  the  filter  with  hot  water  (10 — 12  times)  till  the  wash 
water  ceases  to  give  with  BaCl2  a  test  for  B^SO*.  Collect  the  aque- 
ous filtrate  and  wash-water  and  set  it  aside  to  be  examined  later  for 
glycerin. 

The  funnel  containing  the  washed  fatty  acids  is  now  placed  up- 
right in  a  small  beaker  containing  cold  water,  the  level  of  which 
should  correspond  to  that  of  the  fatty  acid  on  the  filter.  The  fatty 
acids  solidify.  The  product  thus  obtained  is  a  mixture  of  oleic  acid, 
C18H3402,  palmitic  acid  C16H3202,  and  stearic  acid  C18H3602.  Com- 
mercial stearin  which  is  used  in  the  manufacture  of  candles  is  a  mix- 
ture of  palmitic  and  stearic  acids. 

Butter  fat  when  treated  as  above  yields  fatty  acids,  a  part  of 
which  (7  per  cent,  or  more)  are  soluble  in  hot  water;  the  remainder 
(not  over  88  per  cent),  consists  of  insoluble  fatty  acids  and  remains  on 
the  filter.  All  other  animal  fats,  as  lard,  tallow,  etc.,  yield  95.5  per 
cent,  of  insoluble  fatty  acids  (Hehner  and  Angell's  method  for  analysis 
of  butter). 

Reactions  with  Fatty  Acids. 

10. — To  some  of  the  solid  fatty  acid  in  a  test-tube  add  about 
10  c.c.  of  strong  alcohol  and  warm  till  the  acid  dissolves.     Divide  into 


FATS.  13 

three  portions.  To  one  portion  add  a  drop  of  rosolic  acid;  to  another 
portion  1 — 2  drops  of  aqueous  litmus  solution;  to  a  third  portion  a  strip 
of  blue  litmus  paper.  What  is  the  reaction,  and  which  reagent  is 
most  delicate? 

11. — To  a  portion  of  the  fatty  acid  apply  the  test  given  under 
Exp.  5. 

12. — To  a  portion  of  the  fatty  acid  apply  the  test  given  under 
Exp.  6.     What  is  the  result? 

13. — To  about  10  c.c.  of  a  semi-saturated  Na2C03  solution  add  a 
small  portion  of  the  fatty  acids  and  heat.  An  effervescence  results, 
carbonic  acid  is  given  off.  The  fatty  acids  dissolve  and  a  sodium 
soap  is  formed.  Place  the  tube  in  a  beaker  of  cold  water,  a  soap 
jelly  results. 

Warm  the  tube  again  till  the  contents  are  liquid,  then  add  1 — 2 
drops  of  cotton-seed  or  olive  oil  and  shake.  An  opalescent  liquid,  or 
emulsion  forms.  Transfer  a  drop  of  this  to  a  slide,  cover  and  exam- 
ine under  the  microscope.  Note  the  highly  refracting  fat  globules. 
Soap  solutions  emulsify  neutral  fats.  Importance  of  this  fact  in  the 
physiological  absorption  of  fat. 

14. — Place  some  of  the  fatty  acid  in  a  small  beaker,  add  about 
50  c.c.  of  water  and  warm  gently  till  the  fatty  acids  melt.  Then  add 
dilute  NaOH,  drop  by  drop,  and  stir  thoroughly  after  each  addition. 
Continue  addition  of  alkali  till  the  fatty  acid  just  dissolves.  With 
this  sodium  soap  solution  make  the  following  tests: 

(a).     To  some  of  the  solution  add  a  few  drops  of  CaCl2  solution 
An  insoluble   calcium  stearate,  etc.,  forms.      This  calcium  soap  is 
formed  when  hard  water  is  used  with  soap.     What  is  meant  by  hard- 
water? 

(b).  To  another  portion  add  some  lead  acetate  and  warm  gently. 
The  white  sticky  precipitate  which  forms  is  lead  soap.  It  is  known 
and  used  medicinally  as  lead  plaster.  Oleate  of  lead  is  soluble  in 
ether — distinction  from  palmitin  and  stearin. 

15. — Separation  of  Glycerin,  CsH5(OH)s. — The  combined  aqueous  fil- 
trate and  washings  from  the  fatty  acids  (Exp.  9),  should,  if  oily  globules 
are  present,  be  filtered  through  a  wet  filter.  The  filtrate  is  then  care- 
fully neutralized  with  NaOH  and  concentrated  in  an  evaporating 
dish,  first  over  a  flame,  and  finally  on  a  water-bath  almost  to  dryness. 
To  the  residue  add  about  25  c.c.  of  alcohol,  stir  thoroughly  and  allow 
the  mixture  to  stand  for  X — %■  hour,  then  filter.  To  the  residue  add 
another  portion  of  about  15  c.c.  of  alcohol,  stir  well  and  transfer  this 
washing  to  the  filter.     Evaporate  the  alcoholic  filtrate  and  washings, 


14  PHYSIOLOGICAL  CHEMISTRY. 

on  the  water-bath,  to  dryness.  Take  up  the  residue  with  about  15  c.c. 
of  absolute  alcohol  and  transfer  this  entire  mixture  to  a  large  test- 
tube;  then  add  an  equal  volume  of  ether,  cork  and  shake,  and  set 
aside  in  a  beaker  of  cold  water  for  about  a  half  an  hour.  Filter  off 
the  salts  which  are  thus  thrown  out  of  solution  and  cautiously  evapo- 
rate the  alcoholic-ethereal  liquid  on  a  slightly  warmed  water-bath. 
A  syrupy  residue  remains, — glycerin. 

16. — Taste  the  yellowish  syrup  that  is  left. 

17. — Place  a  drop  of  the  residue  on  a  slide  and  add  a  little  pow- 
dered borax.  Then  touch  the  mixture  with  a  platinum  wire  and  place 
this  in  a  Bunsen  flame.     Note  the  green  color. 

18. — Mix  a  drop  or  two  of  the  syrup  with  some  powdered  KHS04 
and  heat  in  a  dry  test-tube.  Compare  the  sesult  with  that  obtained 
in  experiments  6  and  12. 

19. — To  some  glycerin  solution  add  a  little  sodium  hydrate  then 
a  few  drops  of  copper  sulphate.  Instead  of  a  precipitate  a  blue  solu- 
tion results. 

Glycerin  on  treatment  with  a  mixture  of  sulphuric  and 
nitric  acids  yields  an  ester,  03H5(0 .  N02)3,  commonly  known 
as  nitro-glycerin.  When  mixed  with  sand  or  infusorial 
earth  it  is  known  as  dynamite. 


CHAPTER     II. 
CARBOHYDRATES. 

In  this  group  are  usually  placed  those  substances  which 
contain  H  and  O  in  the  same  proportion  as  does  water  (2  : 1) 
and  6  carbon  atoms  or  a  multiple  of  6.  Recent  investiga- 
tions have  shown  that  we  may  have  carbohydrates  contain- 
ing from  4  to  9  or  more  carbon  atoms.  There  are,  further- 
more, unquestionable  sugars,  such  as  rhamnose,  which 
do  not  have  H  and  O  in  the  proportion  of  2  to  1. 

Carbohydrates  are  present  in  comparatively  small 
amounts  in  the  animal  body,  either  free  or  as  constituents 
of  certain  complex  proteids.  They  constitute,  however,  the 
greater  part  of  the  solids  of  plants,  just  as  proteids  make 
up  the  greater  part  of  the  solids  of  the  animal  body.  They 
are  aldehyde  or  ketone  derivatives  of  certain  alcohols. 

The  following  condensed  classification  is  adapted  from 
Tollens: 

I.     Mono- Saccharides  or  Gly coses. 

This  includes  besides  others,  pentoses,  C5H10O5,  and  hexoses, 
C6Hi206,  such  as  dextrose,  laevulose;  and  rhamnose,  06Hi2O5. 

II.     Di-  Saccharides,  or  Saccharoses,  Ci2HMOu. 
Cane-sugar,  milk-sugar,  maltose,  iso-maltose. 

III.  Poly- Saccharides. 

A  few  of  these  compounds  are  crystallizable,  but  most  of  them 
are  amorphous.  The  latter  group  includes  pentosanes,  which  have 
the  same  relation  to  pentoses  as  starch  bears  to  glucose.  Also, 
starch,  and  its  derivatives  amylodextrin,  erythrodextrin,  and  achroo- 
dextrin.     Also  glycogen,  dextran,  and  others;  cellulose. 

IV.  Mannite,  C6HuOe. 

The  compounds  of  this  group  are  related  to  the  true  carbohy- 
drates. 


16  PHYSIOLOGICAL  CHEMISTRY. 

V.     Inosite,  C6Hi206. 

This  is  a  derivative  of  hexamethylene  C6Hi2. 

As  shown  from  the  following-  formulae,  dextrose  or  glu- 
cose contains  an  aldehyde  group,  whereas  laevulose  or 
fructose  contains  a  ketone  group: 

Dextrose  =  CH2OH.CHOH.CHOH.CHOH.CHOH.CHO. 
Laevulose  =  CH2OH .  CHOH .  CHOH .  CHOH .  CO .  CH2OH. 

On  treatment  with  nascent  hydrogen  the  aldehyde  or 
ketone  group  is  readily  reduced  to  the  corresponding  alco- 
hol group  CH2OH,  or  CHOH  yielding  mannite  C6Hu06.  The 
pentoses  in  a  similar  way  yield  corresponding  pentites. 

The  mono-saccharides,  like  aldedydes,  readily  reduce 
salts  of  silver,  copper,  mercury,  etc.  It  should  be  remem- 
bered that  these  salts  are  also  reduced  by  other  sub- 
stances, as  lactose,  maltose,  glucuronic  acid,  "alkapton." 

PENTOSES,  C5H10O5. 

In  plants  there  are  substances,  pentosanes,  which  yield 
on  hydration  pentoses  just  as  starch  on  similar  treatment 
yields  glucose.  A  pentose  has  been  met  with  in  the  decom- 
position of  a  glyco-proteid  obtained  from  the  pancreas.  It 
has  been  found  recently  in  several  urines;  also  in  the  urine 
of  ordinary  and  of  experimental  diabetes. 

The  pentoses  are  strong  reducing  agents,  but  are  not 
fermentable  by  yeasts.  With  phenyl-hydrazin  they  yield 
osazons  which  melt  at  157°— 160°.  On  distillation  with 
hydrochloric  acid  they  yield  furfurol,  which  colors  anilin 
acetate  paper  bright  red. 

HEXOSES,  C6H]206. 

Cane-sugar,  C12H22On,  which  yields  dextrose  and  laevu- 
lose, C6H1206,  on  hydration,  can  therefore  be  considered  as 
an  anhydride  of  these  hexoses.  The  hexoses,  dextrose  and 
laevulose,  are  widely  distributed  in  plants,  especially  in 
acid  fruits;  and  furthermore,  readily  form  on  hydration  of 


CARBOHYDRATES.  17 

starch,  cane-sugar,  glucosides  as  phloridzin,  etc.  Another 
hexose,  galactose,  results  on  hydration  of  lactose,  and  of 
other  carbohydrates,  and  also  of  cerebrin. 

The  three  hexoses  mentioned  are  fermentable  by  yeast. 
On  heating-  with  dilute  mineral  acids  they  yield  laevulinic 
acid,  C5Hs03,  and  humous  substances. 

Dextrose  or  glucose,  also  known  as  grape-sugar  or  starch- 
sugar,  is  formed  during  digestion.  It  is  present  in  small 
amount,  0.1 — 0.2  per  cent.,  in  the  blood;  in  still  less  amount 
in  normal  urine.  In  diabetes  it  is  present  sometimes  in 
considerable  quantities  as  the  characteristic  constituent  of 
urine.  After  the  digestion  of  large  quantities  of  cane- 
sugar,  lactose  or  glucose,  a  reducing  substance  appears  in 
the  urine  (alimentary  glycosuria).  A  part  of  the  cane- 
sugar  may  appear  in  the  urine  as  such.  Glucose  appears 
in  the  urine  after  administration  of  phloridzin,  uranium 
salts,  hydrocyanic  acid;  also  when  the  oxygen  supply  is 
diminished,  and  hence  in  CO  poisoning.  Reducing  sub- 
stances, presumably  glucose,  are  formed  in  the  decomposi- 
tion of  cartilage,  nucleinic  acid,  paranuclein,  nucleoproteid 
of  the  pancreas,  etc. 

It  can  be  obtained  as  minute  crystals  which  are  either 
anhydrous  or  contain  one  molecule  of  water.  It  is  only 
about  3 — 5  as  sweet  as  cane-sugar.  It  is  soluble  in  about  an 
equal  part  of  water;  insoluble  in  absolute  alcohol.  The 
solutions  are  dextro-rotatory.  The  melting-point  is  at 
144°— 146°;  above  200°  caramel  forms. 

Glucose. 

In  the  following  experiments,  unless  otherwise  indi- 
cated, a  two  per  cent,  solution  of  glucose  is  employed: 

1. — Moliseh's  Reaction. — To  about  K  c.c.  of  the  dilute  sugar  solu- 
tion add  1 — 2  drops  of  an  alcoholic  solution  (15  per  cent.)  of  a-naphthol. 
Then  add  slowly  about  1  c.c.  of  concentrated  kH2S04  so  that  it  runs 
down  the  side  of  the  inclined  tube  and  forms  a  layer.  A  beautiful 
reddish  violet  ring-  forms  at  the  zone  of  contact. 


18  PHYSIOLOGICAL   CHEMISTRY. 

This  is  a  general  reaction  due  to  the  formation  of  fur- 
furol  and  is  given  by  all  carbohydrates.  Apply  this  test  to 
some  normal  urine;  and  to  urine  diluted  with  5  parts  of 
water.  If  the  test  is  given  by  the  latter  it  indicates  that 
the  carbohydrates  of  the  urine  are  increased. 

2. — Place  some  of  the  dry  glucose  in  a  tube  and  heat  gently  over 
a  flame.  It  melts,  then  turns  yellow  and  finally  dark-brown.  The 
peculiar  odor  is  that  of  burnt-sugar.  Allow  the  tube  to  cool,  then  add 
water  and  warm  slightly.  Note  the  dark-yellow  or  brownish  color  of 
the  solution.  Caramel  is  a  harmless  coloring  matter  and  is  employed 
extensively  for  coloring  liquors,  vinegars,  etc. 

3. — To  some  dry  glucose  add  cold,  concentrated  H2S04  and  let 
stand.  The  liquid  remains  colorless,  or  at  most  is  light  yellow.  Dis- 
tinction from  cane-sugar.  See  experiment  3  under  cane-sugar.  After 
comparing  this  with  the  corresponding  experiment  with  cane-sugar, 
gently  heat  the  glucose  tube.  It  promptly  blackens,  due  to  humin 
substances.     Laevulinic  acid  is  formed  at  the  same  time. 

4. — To  the  sugar  golution  add  some  strong  KOH  solution  and 
heat.  The  liquid  becomes  yellow,  then  dark-brown.  The  sugar  read- 
ily undergoes  oxidation  in  alkaline  solution.  With  solid  KOH,  be- 
cause of  the  heat  generated,  the  reaction  is  sometimes  violent. 

This  test  when  applied  to  urine  is  known  as  Moore- 
Heller's  test.  In  that  case  the  precipitate  that  forms  is 
due  to  earthy  phosphates.  The  test  is  not  particularly 
delicate  and  is  certainly  not  reliable  since  other  substances 
may  yield  dark  solutions  under  similar  conditions,  namely, 
alkapton,  lactose,  maltose,  etc. 

Compare  this  reaction  with  experiment  4  under  cane- 
sugar. 

5. — To  the  sugar  solution  add  y2  volume  of  Na2C03  solution,  then 
1 — 2  drops  of  a  freshly  prepared  solution  of  potassium  f erricyanide,  and 
boil.  The  liquid  becomes  colorless — due  to  a  reduction  of  the  salt  to 
a  ferrocyanide. 

6. — To  the  sugar  solution  add  a  little  ammoniacal  silver  nitrate 
and  a  few  drops  of  KOH  and  warm  gently.     A  mirror  of  metallic  sil- 


CARBOHYDRATES.  19 

ver  forms,  especially  if  the  solutions  are  dilute.     The  silver  has  been 
reduced. 

The  ammoniacal  silver  nitrate  is  prepared  by  adding 
ammonium  hydrate  to  silver  nitrate  till  the  precipitate  just 
disappears. 

7. — To  the  sugar  solution  add  one  drop  of  a  freshly  prepared 
solution  of  sodium  indigo  sulphate,  also  add  a  little  Na2C03  solution 
and  heat.  The  blue  color  changes  first  to  violet,  then  to  red,  yellow, 
and  finally  the  liquid  is  colorless.  The  indigo  has  been  reduced  to 
indigo-white.  Cool  the  tube  under  the  hydrant  and  shake.  The 
indigo-white  is  oxidized  to  indigo-blue.  On  heating  the  blue  is  again 
reduced.  Litmus  and  other  coloring  agents  are  reduced  in  a  similar 
manner. 

The  following-  reactions  should  be  applied,  side  by 
side,  to  the  aqueous  solution  of  glucose  and  to  diabetic 
urine,  or  to  urine  containing  about  one  per  cent,  of  sugar. 

8. — Trammer's  Test. — Render  the  solution  or  urine  strongly  alka- 
line with  KOH  and  boil,  then  add  a  few  drops  of  copper  sulphate  solu- 
tion and  warm  again;  a  reddish-yellow  precipitate  of  cuprous  oxide 
forms.  If  excess  of  copper  be  added,  the  copper  hydrate  precipitate 
will  mask  small  amounts  of  the  red  precipitate.  If  too  little  copper 
has  been  added,  a  white  precipitate  of  uric  acid  and  nuclein  bases 
(alloxuric  bodies)  may  form. 

8c<. — FehUny's  Test. — Boil  some  Fehling's  solution  in  a  test-tube 
and  then  add  the  sugar  solution,  or  the  suspected  urine,  and  boil. 
Cuprous  oxide  is  thrown  down. 

The  urine  if  strongly  acid  should  be  rendered  alkaline. 
This  is  the  test  commonly  employed  when  examining  urine 
for  sugar.  It  should  be  remembered  that  it  is  not  an  abso- 
lute test,  since  the  urine,  in  rare  cases,  may  contain  other 
reducing  substances  (alkapton).  A  small  amount  of  sugar 
may,  moreover,  escape  detection,  since  the  cuprous  oxide 
may  be  held  in  solution  by  creatinin  and  other  urine  con- 
stituents. 


20  PHYSIOLOGICAL  CHEMISTRY. 

It  should  furthermore  be  remembered  that  Fehling's 
solution  deteriorates  on  keeping-,  so  that  on  heating-  the 
solution  itself,  a  red  precipitate  of  cuprous  oxide  may  form. 
It  is  advisable,  therefore,  to  keep  the  two  constituents  of 
Fehling's  solution  in  separate  bottles  and  to  mix  equal  vol- 
umes just  before  use. 

Pavy's  solution,  employed  for  the  same  purpose,  is  a 
solution  of  copper  hydrate  in  ammonium  chloride. 

9. — To  some  Barfoecfs  solution  add  some  glucose  solution  and  boil. 
The  cuprous  oxide  precipitate  forms.  Milk-sugar,  cane-sugar,  maltose 
and  dextrin  do  not  reduce  this  solution. 

Barf oed's  reagent  is  an  acid  solution  of  copper.  It  is 
prepared  by  dissolving  one  part  of  copper  acetate  in  15 
parts  of  water.  To  200  c.c.  of  this  solution,  5  c.c.  of  a  38 
per  cent,  acetic  acid  solution  are  added. 

10. — Boettger\s  Test. — Render  the  specimen  alkaline  with  sodium 
or  potassium  hydrate,  then  add  a  minute  quantity  of  basic  bismuth 
nitrate  and  heat — a  black  color  or  precipitate,  due  to  reduced  bis- 
muth, forms.  Albumin  if  present  must  be  removed.  Owing  to  the 
action  of  the  alkali  on  the  sugar  the  solution  may  color.  This  can  be 
avoided  by  substituting  sodium  carbonate  for  the  alkali. 

10a. — JSfylander's  Test. — Dissolve  10.33  g.  of  'sodium  hydrate  in 
100  c.c.  of  water;  add  2  g.  of  basic  bismuth  nitrate,  and  4  g.  Rochelle 
salts;  warm  and  filter.  This  reagent  keeps  better  than  Fehling's 
solution. 

To  10  volumes  of  the  sugar  solution  or  urine  add  one  volume  of 
the  reagent  and  boil  2 — 3  minutes.     Then  let  stand  for  10 — 15  minutes. 

Concentrated  urines  may  become  blackish  with  this 
reagent;  this  may  also  occur  if  chrysophanic  acid  is  pres- 
ent in  the  urine.  On  the  other  hand  the  reaction  is  more 
delicate  than  Fehling's  solution,  where,  as  pointed  out, 
small  amounts  of  cuprous  oxide  may  be  held  in  solution. 

Alkaline  solutions  of  mercury  salts  are  also  employed 
in  testing  for  glucose  (Knapp,  Sachsse). 


CARBOHYDRATES.  21 

11.  —  Phenyl-hydrazin  Test.  —  Phenyl-hydrazin  on  heating  with 
sugar  forms  phenyl-glucosazon,  C6H10O*  (N2H.C6H5)2.  This  forms  bun- 
dles of  yellow  needles  which  melt  at  204-205°. 

C6H12Os  +  2C6H5.N2H3  =  C18H22N404  +  2H20  +  2H. 

Application  to  the  urine. — Place  in  a  small  beaker  about  50  c.c. 
of  the  clear  urine  add  1 — 2  g.  of  phenyl-hydrazin  hydrochloride  and 
about  2 — 4  g.  of  sodium  acetate,  cover  with  a  watch-glass  and  warm  on 
the  water-bath  for  l/2 — 1  hour,  then  turn  off  the  light  and  allow  the 
solution  to  cool  on  the  water-bath.  Examine  under  the  microscope 
the  deposit  which  forms.  If  amorphous,  or  if  it  is  desirable  to  purify 
the  crystals,  dissolve  on  the  filter  in  hot  alcohol.  To  the  nitrate  add 
water  and  boil  till  the  alcohol  is  expelled — on  cooling,  the  character- 
istic yellow  crystals  appear.  Filter,  wash,  dry  and  determine  the 
melting-point  (see  urea). 

The  phenyl-hydrazin  reaction  with  sugars  is  of  very 
great  importance  in  their  identification.  It  forms  with 
sugars,  when  heated  sufficiently  long-  on  the  water-bath, 
osazones.  The  various  sugars  yield,  therefore,  correspond 
ing  osazones,  which  are  yellowish,  and  differ  in  crystalline 
form,  melting-point,  solubility,  and  optical  behavior.  The 
determination  of  the  melting-point  is  especially  valuable. 

12. — Fermentation  Test — Rub  up  some  of  the  solution  or  of  the  sus- 
pected urine  with  a  little  yeast.  Fill  the  mixture  into  a  large,  wide 
test-tube  provided  with  a  perforated  stopper  through  which  passes  a 
tube  bent  into  a  U  shape — the  free  arm  being  longer  than  the  one 
that  passes  through  the  cork.  Care  should  be  taken  to  fill  the  tube 
so  that  no  air  is  present  in  the  test-tube  when  it  is  inverted.  Set  the 
tube  aside  in  an  inverted  position  in  a  warm  place  for  24  hours  and 
observe  the  accumulation  of  gas — fermentation. 

When  the  fermentation  is  completed  place  the  tube  in  an  up- 
right position  in  a  dish  of  water,  remove  the  stopper  and  by  means  of 
a  bent  pipette  introduce  a  little  potassium  hydrate  solution.  What 
is  the  result  ? 

C6H1206  =  2  C2H5OH  +  2  C02. 

Under  the  influence  of  certain  bacteria,  glucose  readily  under- 
goes lactic  acid,  butyric  acid,  or  viscous  fermentation. 


22  PHYSIOLOGICAL  CHEMISTRY. 

Laevulose,  also  known  as  fruit-sugar  or  fructose,  occurs 
widely  distributed  in  the  plant  kingdom.  It  is  also  present 
with  dextrose  in  honey.  While  starch  on  hydration  yields 
dextrose,  there  are  analogous  substances,  as  inulin,  C6H10O5, 
which  on  similar  decomposition  yield  laevulose.  In  excep- 
tional cases  it  has  been  met  with  in  the  urine  of  diabetes. 
When  administered  in  diabetes  a  part  may  be  changed  to 
glucose  and  to  glycogen,  and  a  part  may  be  eliminated  as 
such  (Haycraft). 

This  sugar  crystallizes  with  great  difficulty  and  for  that 
reason  it  is  ordinarily  met  with  as  a  thin  syrup.  It  is  read- 
ily soluble  in  water,  insoluble  in  cold  absolute  alcohol. 
The  solutions  are  laevo-rotatory. 

The  rotation  is  greater,  and  in  opposite  direction,  than 
that  of  cane-sugar.  Hence  on  hydration  of  cane-sugar  the 
resulting  mixture  is  laevo-rotatory,  and  is  therefore  called 
invert-sugar.  Inversion,  as  applied  to  complex  carbohy- 
drates, is  synonymous  with  hydration. 

Like  glucose,  it  readily  reduces  metallic  oxides;  is  fer- 
mented by  yeast  and  forms  the  same  osazon. 

Galactose  is  formed  with  dextrose  on  hydration  of  milk- 
sugar,  and  other  carbohydrates;  also  of  cerebrin.  In  yellow 
lupine  a  compound,  galactite  (Ritthausen),is  present  which 
yields  galactose  on  hydration.  It  crystallizes  in  needles 
or  plates  which  melt  at  168°.  It  is  dextro-rotatory.  It 
reduces  Fehling's  solution  and  is  said  to  ferment  with 
yeast.  It  forms  an  osazone  which  melts  at  193°.  On  oxid- 
ation it  yields  mucic  acid — distinction  from  dextrose.  The 
origin  of  galactose  as  a  constituent  of  milk-sugar  is  not 
known.  It  may  be  derived  from  antecedents  in  the  plant 
food,  and  on  the  other  hand  may  be  formed  from  glycogen 
or  even  glucose  in  the  body. 

CANE  SUGAR,  C12H22On. 

Saccharose,  Sucrose. — This  sugar  is  widely  distributed  in 


CARBOHYDRATES.  23 

plants  in  the  leaves  of  which  it  is  formed,  under  the  influ- 
ence of  light  and  possibly  of  chlorophyll.  It  is  then  trans- 
ported to  different  parts  of  the  plant  and  may  be  stored  up 
in  the  roots  as  in  the  case  of  beet-root,  or  in  the  stalk,  as 
in  sugar  cane.  In  acid  liquid  it  very  readily  undergoes 
inversion  and  for  that  reason  it  is  not  present  in  strongly 
acid  fruit  juices  but  is  represented  there  by  dextrose  and 
laevulose.  In  moderately  acid  fruits  as  nuts,  apples,  mel- 
ons, bananas,  sweet  oranges,  it  is  present  as  such  with 
more  or  less  glucose.  The  cane  sugar  which  is  removed 
from  the  flower  by  the  bee  becomes  almost  wholly  inverted 
when  made  into  honey. 

It  forms  large  monoclinic  crystals  which  dissolve  in  1.5 
parts  of  water  at  20°.  The  solution  is  strongly  dextro- 
rotatory. It  melts  at  160°  and  on  further  heating  it  yields 
caramel.  It  is  decomposed  by  dilute  acids,  slowly  in  the 
cold,  very  rapidly  on  heating.     The  change  is  as  follows; 


C12H220„  +  H20  —  C6Hi206  -+-  C6H1206 

Dextrose.  Laevulose 


This  hydration  is  also  brought  about  by  many  ferments, 
such  as  the  invertin  of  yeast;  by  bacteria  and  moulds; 
also  by  the  acid  gastric  juice  but  not  by  the  pancreas. 
Once  inverted  the  resultant  invert-sugar  is  readily  subject 
to  various  fermentations  such  as  alcoholic,  viscous,  lactic 
acid,  etc. 

Before  inversion  it  is  strongly  dextro-rotatory  and  does 
not  reduce  Fehling's  solution.  After  inversion  it  is  less  dex- 
tro-rotatory, or  even  laevo-rotatory,  and  reduces  Fehling's 
solution.  With  phenyl-hydrazin  it  does  not  form  a  corres- 
ponding osazon,  but  does  form,  owing  to  inversion,  the 
phenyl-glucosazon.  This  behavior  and  the  non-reduction 
of  metallic  oxides  distinguishes  cane-sugar  from  maltose 
and  lactose.  The  latter,  therefore,  still  show  the  aldehyde 
character. 


24  PHYSIOLOGICAL   CHEMISTRY. 

Apply  the  following-  reactions,  with  the  exception  of  2  and  3,  to  a 
2  per  cent,  aqueous  solution  of  cane-sugar,  and  compare  these,  side 
by  side,  with  the  corresponding"  reactions  of  glucose: 

1. — Molisch's  Reaction. — Apply  as  given  in  Exp.  1,  under  glucose 

2. — Caramel  Reaction. — Apply  as  given  in  Exp.  2,  under  glucose. 

3. — Sulphuric  Acid  Reaction. — Apply  as  given  in  Exp.  3,  under 
glucose.  The  cold  acid  in  a  few  minutes  colors  yellow,  then  becomes 
black — distinction  from  dextrose.     Humin  substances  are  formed. 

4. — Potassium  Hydrate  Reaction. — Apply  as  given  in  correspond- 
ing test  under  glucose  and  carefully  note  the  difference. 

5. — Apply  Fehling's  solution  as  in  Exp.  8a  under  glucose. 

6. — Test  with  Barfoed's  reagent,  as  in  Exp.  9  for  glucose. 

7. — Test  with  Nylander's  reagent,  as  in  Exp.  10a  under  glucose. 

8. — Apply  the  fermentation  test  as  in  Exp.  12  under  glucose,  and 
compare  the  rapidity  of  fermentation  with  that  of  glucose. 

9. — Place  50  c.c.  of  the  cane-sugar  solution  in  a  small  beaker,  add 
6 — 8  drops  of  concentrated  HC1  and  boil  for  2 — 3  minutes.  Then  cool 
render  alkaline  with  sodium  or  potassium  hydrate.  To  this  solution 
now  apply  tests  4,  5,  6,  7,  as  given  above.     Note  the  results. 

Lactose,  C,2H22On  +  H20. 

Lactose,  or  milk-sugar,  occurs  probably  in  the  milk  of 
all  animals.  The  amount  present  varies  from  3 — 5 — 6  per 
cent.  It  has  been  found  in  the  urine  during-  the  later  stages 
of  pregnancy  and  immediately  after  birth.  It  is  said  to 
occur  in  one  plant. 

It  forms  large  rhombic  crystals  which  are  soluble  in  6 
parts  of  cold  water  and  2^  parts  of  boiling  water.  The 
solution  is  dextro-rotatory.  When  heated  to  170 — 180°  it 
forms  a  lacto-caramel,  C6H10O5;  melts  at  203.5°.  On  heat- 
ing with  acids,  hydration  takes  place  according  to  the 
equation: 

C12H22On  +  H20  =  C6H1206  +  C6H]206. 

Galactose.  Dextrose. 

On  further  heating  with  acids,  humin  and  formic  and  laev- 
ulinic  acids  form.  On  oxidation  with  nitric  acid,  inversion 
first  takes  place  as  above,  and  then  the  galactose  is  oxi- 


CARBOHYDRATES.  25 

dized  to  mucic  acid,  whereas  the  dextrose  forms  saccharic 
acid.  It  reduces  Fehling's  solution  but  is  only  2/i  as  strong 
as  dextrose.  Unlike  the  latter,  it  is  not  fermented  by 
yeast.  Bacteria  readily  bring-  about  lactic  acid  f ermenta 
tion.  In  kephir  and  kumyss  the  sugar  is  changed  to  alco- 
hol and  lactic  acid. 

With  phenyl-hydrazin  it  combines  to  form  a  lactosazon 
which  crystallizes  on  cooling  as  round  aggregates  of  yellow 
needles  which  melt  at  200°.  Its  behavior  to  cold  concen 
trated  sulphuric  acid  and  to  alkalis  also  serves  to  distin- 
guish it  from  dextrose  and  cane-sugar  respectively.  Alkalis 
yield  lactic  acid  and  pyrocatechin. 

To  a  two  per  cent,  solution  of  lactose  apply  the  tests  1 — 8  as  given 
under  cane-sugar.  For  the  preparation  of  milk-sugar  see  Milk.  Lac- 
tose is  estimated  according  to  the  method  given  under  Milk  analysis 
(Chapter  XI). 

Maltose,  C12H22On  +  H20. 

This  sugar  is  formed  by  the  action  of  the  ferment  dias- 
tase, contained  in  malt  or  sprouting  barley,  on  starch.  It  is 
also  formed  by  the  ferments  of  the  saliva,  pancreas  and 
liver.  The  formation  of  dextrin  precedes  that  of  maltose. 
When  starch  is  heated  with  H2S04,  maltose  is  temporarily 
produced.  Consequently  crude  glucose  and  glucose  syrup 
will  contain  maltose  in  small  amounts. 

It  forms  fine  white  needles,  grouped  in  little  masses. 
It  is  soluble  in  water  and  in  dilute  alcohol.  On  oxidation 
with  nitric  acid  it  yields  saccharic  acid.  On  heating  with 
sulphuric  acid  it  yields  two  molecules  of  dextrose.  This 
change  is  also  accomplished  by  ferments. 

C12H,A,  +  H20  =  C6H,A  +  C.HwO,. 

Like  dextrose  it  is  easily  fermented  by  yeast,  and  readily 
reacts  vvitli  potassium  hydrate,  and  with  Pehling's  solution. 
Lt  reduces  the  latter  more  weakly  than  does  dextrose;    10 


26  PHYSIOLOGICAL,    CHEMISTRY. 

c.c.  of  Fehling's  solution  represents  77.8  mg.  maltose.  The 
reaction  with  Barfoed's  reagent  serves  to  distinguish  it  from 
dextrose.  With  phenyl -hydrazin  it  forms  an  osazon — malt- 
osazon.  This  forms  yellow  separate  needles,  which  melt  at 
206°.  It  dissolves,  or  can  hold  in  solution,  ferric  hydrate. 
It  is  dextro-rotatory. 

Iso-Maltose,  is  an  isomer  of  maltose.  It  is  amorphous 
and  is  formed  by  the  action  of  acids  and  ferments  on  starch. 
It  has  been  prepared  synthetically  from  glucose  by  the 
action  of  concentrated  HC1.  Unlike  maltose,  it  is  more 
difficultly  fermentable,  and  forms  an  osazon  which  melts  at 
153°  It  readily  reduces  Fehling's  solution.  It  is  converted 
by  diastase  into  maltose. 

The  relation  of  the  three  di-saccharides  can  be  seen 
from  the  following: 

Cane-sugar  -+-  HsO  =  glucose  -j-  laevulose. 
Milk-sugar  -4-  H20  =  glucose  +  galactose. 
Maltose  +  H2O  =  glucose  +  glucose. 

1. — To  100  c.c.  of  boiling  water  add  10  g.  of  starch  and  stir  till  an 
even  starch  paste  forms.  Then  cool  to  60°  and  add  1  g.  of  powdered 
malt,  immerse  in  a  water-bath  at  60°  for  one  hour.  At  inter- 
vals of  10  minutes  test  1 — 2  c.c.  of  the  liquid  with  iodine  for  dextrin 
(see  page  29)  Then  boil  and  filter.  Evaporate  }4  of  the  filtrate  to  a 
thick  syrup  and  set  this  aside  for  several  days  to  crystallize.  The 
addition  of  a  thread,  or  of  a  crystal  of  maltose  will  favor  crystalliza- 
tion.    Note  the  taste  of  the  syrup. 

To  the  remaining  )4  of  the  filtrate  apply  the  tests  4 — 8  inclusive 
as  given  under  cane-sugar. 

Starch,   (C6H10O5)n . 

Starch,  or  amylum,  is  a  highly  complex  carbohydrate 
and  the  value  of  n  in  the  above  formula  is  not  determined. 
It  is  placed  by  some  at  as  high  as  200.  Starches  are  also 
known  as  glucosins,  since  on  hydration  they  yield  as  a  final 
product  glucose  or  dextrose,  whereas  the  inulins  or  laevu- 
lins,    which   correspond    to   starch,    yield  laevulose.     The 


CARBOHYDRATES.  27 

inulins  are  comparatively  rare,  whereas  starch  is  a  most 
widely  distributed  plant  constituent.  It  is  evidently  formed 
from  CO.,  by  chlorophyll  in  the  presence  of  water.  In  plants 
the  excess  of  sugar  is  stored  up  as  starch,  while  in  animals 
it  is  stored  up  as  glycogen.  In  the  body  of  animals  starch 
can  unquestionably  be  converted  into  and  deposited  as  fat. 
It  is  known  that  bacteria  acting  on  starch  can  give  rise  to 
certain  fatty  acids. 

Starch  is  contained  in  the  so-called  starch-granules, 
which  have  a  characteristic  appearance  and  can  be  readily 
recognized  under  the  microscope.  The  form  of  the  granules 
as  obtained  from  one  plant  differs  from  that  obtained  from 
other  plants.  The  size  of  the  granules  varies  greatly  even 
in  starch  of  the  same  variety.  The  starch  proper  is  depos- 
ited in  these  granules  in  layers  around  one  or  more  nuclei. 
Some  cellulose  is  present.  Frequently,  as  a  result,  concen- 
tric rings  will  be  observed  in  the  starch  granule. 

On  heating  to  150 — 170°  it  becomes  yellowish,  and 
also  soluble  in  water;  that  is,  dextrin  is  formed.  Com- 
mercial dextrin,  which  is  used  extensively  as  a  mucilage,  is 
prepared  in  this  way. 

Starch  is  insoluble  in  cold  water.  In  the  presence  of 
chloride  of  zinc  and  other  salts  it  swells  up  and  dissolves. 
On  heating  with  water  to  60 — 70°  it  swells  to  a  paste  but 
does  not  form  a  true  solution.  At  a  higher  temperature  it 
does  dissolve,  forming  soluble  starch  and  hydrolytic  pro- 
ducts described  below.  With  glycerin,  especially  on  heat- 
ing, it  forms  soluble  starch.  On  heating  with  dilute  acids 
it  dissolves  readily,  or  is  rather  hydrated,  forming  soluble 
products.  The  final  product  of  the  action  of  an  acid  is 
dextrose.  HC1  acts  more  rapidly  than  H2S04.  Diastatic 
enzymes,  such  as  are  contained  in  malt,  saliva,  pancreas, 
dissolve  starch,  forming  a  number  of  intermediate  products 
and  finally  maltose,  not  dextrose.  Starch  is  not  fermented 
by  yeast  but  is  atfected  by  bacteria,  such  as  lactic  acid 
and  butyric  acid  bacilli;  also  by  moulds.     Nitric  acid   tirsl 


28 


PHYSIOLOGICAL    CHEMISTRY. 


inverts  starch,  then  oxidizes;   the  products  are  saccharic, 
tartaric,  and  oxalic  acids. 

The  products  formed  by  the  hydration  of  starch,  brought 
about  by  water  under  pressure,  by  acids,  or  by  ferments, 
are  presented  in  the  following-  table: 


Starch 

Soluble  Starch.... 
Erythrodextrin  . . 
Achroodextrin . . . 

Maltodextrin 

Isomaltose 

Maltose 

Dextrose 


Iodine, 

blue 

tt 

red. 

c< 

0. 

a 

0. 

a 

0. 

a 

0. 

a 

0. 

Fehling, 


0 

0 

+  very  slight. 

-j-  slight. 

+ 

+  Barfoed,  0. 


Tasteless 


Sweetish. 
Sweet. 


1. — Examine  microscopically  and  sketch  the  granules  of  the  fol- 
lowing starches:  potato,  wheat,  buckwheat,  corn,  arrowroot  and  rice. 
Note  the  shape  of  granules,  the  number  of  rings  if  any,  and  the  cleft 
or  hilum. 

2. — Place  a  little  starch  in  a  test-tube,  add  water  and  shake 
thoroughly,  then  filter.  To  the  filtrate  add  a  drop  of  iodine  solution^ 
No  color  is  formed,  since  starch  is  insoluble.  Add  a  drop  or  two  of 
iodine  to  the  residue  on  the  filter — a  blue  color  results. 

.3.— Soluble  Starch.— Place  100  c.c.  of  water  in  a  beaker  and  boil, 
then  add  1  g.  of  powdered  starch  and  continue  boiling  for  2—3  minutes, 
stirring  constantly.     A  starch  paste  forms. 

4.— Place  some  of  the  starch  solution  obtained  in  experiment  3  in 
a  tube  and  add  a  drop  of  iodine  solution.  A  deep-blue  color  results 
Now  heat  the  contents  of  the  tube;  the  color  disappears,  to  reappear 
on  cooling.  The  blue  color  is  due  to  the  so-called  starch  iodide,  which 
possesses  a  variable  composition. 

5. — To  some  of  the  starch  solution  add  an  excess  of  tannic  acid. 
A  yellowish  white  precipitate  forms  or  the  liquid  becomes  highly 
opaque. 


6.— Boil  some  of  the  starch  solution  with  Fehling's  solution, 
reaction  takes  place. 


No 


7.— To  about  50  c.c.  of  the  starch  solution  in  a  beaker  add  y%  c.c. 
of  H2S04,  cover  with  a  watch-glass  and  boil  for  15  minutes.  Replace 
the  water  that  may  be  lost  by  evaporation.     Now  place  some  of  the 


CARBOHYDRATES.  29 

liquid  in  a  tube,  render  alkaline  with  sodium  or  potassium  hydrate, 
add  some  Fehling's  solution  and  boil.  If  no  reduction  takes  place, 
continue  heating  the  contents  of  the  beaker  for  another  15  minutes 
and  test  as  before.  Inversion  has  taken  place:  a  reducing  sugar  (glu- 
cose) has  been  formed,  as  in  the  similar  experiment  with  cane-sugar4 
This  experiment  is  the  basis  of  the  commercial  manufacture  of 
glucose. 

Dextrin,  (C6H10O5)n  . 

As  explained  above,  a  number  of  compounds  are 
included  under  this  head.  They  are  the  first  hydration 
products  of  starch.  The  commercial  dextrin  is  prepared 
by  heating  starch  to  150 — 160°  with  or  without  water;  also 
by  drying-  starch  at  100°  previousl}T  suspended  in  very 
dilute  nitric  acid;  or  by  treatment  with  acids  or  malt  and 
subsequent  precipitation  with  alcohol.  The  behavior  'of 
the  several  A^arieties  of  dextrin  to  iodine  has  been  indi- 
cated above  and  will  be  demonstrated  in  connection  with 
the  work  on  saliva. 

Unlike  starch,  dextrin  is  very  readily  soluble  in  water. 
The  solution  is  not  fermented  by  yeast,  but  must  first  be 
changed  by  hydration  to  maltose.  Test  a  one  per  cent,  solu- 
tion as  follows: 

* 
1. — To  some  of  the  solution  add  tannic  acid — no  precipitate;  dis 
tinction  from  starch,  gelatin,  albumin. 

2. — To  some  of  the  solution  add  a  drop  or  two  of  iodine  solution. 
What  isjthe  result  ?    And  to  what  is  it  due '? 

3. — To  some  of  the  solution  add  Fehling's  solution  and  boil.  Or- 
dinary dextrin  contains  more  or  less  of  reducing  substances.  If  pure, 
no  reaction  would  take  place. 

Glycogen,  (C6H10O5)n  . 

This  carbohydrate  was  first  discovered  in  the  liver  and 
has  since  been  shown  to  be  present,  in  greater  or  less 
amount,  in  all    the  tissues  of  the  anirral  body.    The  an.oim 


30  PHYSIOLOGICAL    CHEMISTRY. 

of  glycogen  in  the  liver  will  vary  according  to  the  food. 
Ordinarily  it  constitutes  from  1 — 4  per  cent,  but  may,  after 
a  rich  carbohydrate  diet,  amount  to  12 — 16  per  cent.  In 
fresh  muscle  it  amounts  to  about  0.6  per  cent.,  and  disap- 
pears from  the  muscle  as  a  result  of  work  or  starvation.  It 
is  present  in  Liebig's  meat  extract  (1 — 1.5  percent.).  Al- 
though present  in  small  amounts  in  normal  blood,  it  is  con- 
siderably increased  after  extirpation  of  the  pancreas.  It 
is  present  in  larger  amount  in  pus,  and  in  leucocytes. 
Undoubtedly  it  is  a  constituent  of  all  living  animal 
cells.  It  is  abundant  in  embryonic  tissue,  and  the  liver  of 
a  new-born  dog  has  been  found  to  contain  as  much  as  11 
per  cent.  It  is  present  in  considerable  quantity  in  mol- 
luscs, notably  in  oysters.  It  has  been  found  in  certain 
plants;  fungi,  as  truffles;  also  in  mucor  and  in  the  yeast. 

Glycogen  is  related  to  dextrin  and  to  amylodextrin  or 
soluble  starch.  It  has  the  same  percentage  composition  as 
starch  or  dextrin.  The  exact  formula  is  not  known.  It 
would  seem  that  the  multiple  n  in  the  above  formula  is  6, 
although  some  place  it  at  10.  Glycogen  is  derived  from 
the  food,  more  especially  the  carbohydrates.  The  excess 
of  carbohydrates,  whether  starch  or  various  sugars,  is 
promptly  stored  up  in  the  liver  to  be  given  off  under  the 
influence  of  ferments  according  to  the  need  of  the  body. 
Exclusively  proteid  diet  likewise  gives  rise  to  glycogen. 

Various  diastatic  ferments,  such  as  are  present  in  malt, 
saliva,  pancreas,  blood,  liver,  etc.,  invert  glycogen.  The 
change  is  similar  to  that  which  starch  or  soluble  starch 
undergoes  under  like  conditions.  That  is  to  say,  erythro- 
dextrins,  achroodextrin,  isomaltose,  maltose,  and  event- 
ally  glucose  form.  On  heating  with  water  at  high 
temperature,  or  with  dilute  acids,  a  similar  hydration 
results.  Owing  to  the  action  of  these  ferments  of  the 
body,  it  follows  that  in  dead  liver  or  muscle  the  amount 
of  glycogen  rapidly  decreases,  and  is  replaced  by  a  dex- 
trin body,  or  by  maltose  or  glucose. 


CARBOHYDRATES.  31 

The  following  table  shows  the  relation  of  glycogen  to 
sugar  in  the  liver  of  a  rabbit  at  different  periods  after 
death  (Girard): 


10  min. 


Sugar,  per  cent 0.75 

Glycogen,  per  cent I   9 .  56 


24  hrs. 


3.58 
6.35 


48  hrs. 


3.85 

4.28 


The  action  of  the  diastatic  ferments  is  most  marked  in 
neutral  or  very  slightly  acid  solutions.  A  one  per  cent,  solu- 
tion of  sodium  carbonate  inhibits  the  change  and  so  does 
the  acidity  of  a  solution  of  C02.  It  is  possible  that  the  car- 
bonic acid  prevents  or  retards  the  hydration  of  glycogen  in 
the  body.     Glycogen  is  not  affected  by  yeast. 

Glycogen  is  an  amorphous,  white,  tasteless  powder 
which  dissolves  in  warm  water  to  form  an  opalescent  liquid. 
The  opalescence  disappears  on  the  addition  of  an  acid  or 
alkali.  On  the  addition  of  iodine  the  solution  becomes  red 
or  brown  (erythrodextrin).  The  color,  like  that  of  starch 
iodide,  disappears  on  heating.  The  solutions  are  strongly 
dextro-rotatory.  It  is  precipitated  from  impure  solution 
by  alcohol. 

1. — Isolation  of  Glycogen. — The  following-  method  gives  the  best 
results.  It  may  be  applied  to  50  g.  of  fresh  liver,  or  to  %  pint  of  oys- 
ters. The  material  is  cut  up  as  fine  as  possible.  If  liver  is  used  it 
can  be  put  through  a  sausage  machine.  To  the  material  then  add  10 
parts  of  boiling  water  slightly  acidulated  with  acetic  acid.  Strain 
the  opalescent  liquid  through  muslin.  This  liquid  contains,  besides 
glycogen,  some  proteids  and  gelatin.  To  remove  the  latter,  first  con- 
centrate to  a  small  volume,  then  add  alternately  a  few  drops  of  HC1 
and  of  potassium  mercuric  iodide  till  a  precipitate  ceases  to  form. 
Finally  filter  off  a  little  of  the  liquid  and  test  it  with  acid  and  reagent 
to  make  sure  that  all  the  proteids  are  precipitated.  If  this  is  the 
case,  strain  the  liquid  through  muslin,  then  filter  through  paper,  and 
to  the  filtrate  add  two  volumes  of  alcohol  and  stir  thoroughly.  Allow 
the  glycogen  to  settle,  then  filter  off,  wash  with  dilute  alcohol  (2  parts 
alcohol  to  1  part  water).  Finally  transfer  to  a  beaker,  cover  with 
absolute  alcohol  and  let  stand  an  hour  or  more.     Then  filter  off  the 


32  PHYSIOLOGICAL  CHEMISTRY. 

glycogen,  fold  the  filter  and  gently  squeeze  off  excess  of  alcohol, 
finally  press  between  several  layers  of  filter  paper  till  dry.  Powder, 
if  necessary. 

The  reagent  employed  above  is  prepared  by  adding  mercuric 
iodide  to  a  warmed  5  per  cent,  solution  of  KI  till  it  ceases  to  dissolve. 
The  liquid  is  then  cooled  and  filtered. 

With  glycogen  isolated  as  above  make  the  following' 

tests: 

1. — To  some  glycogen  in  a  small  beaker  add  20 — 30  c.c.  of  water 
and  warm.  The  glycogen  dissolves,  forming  an  opalescent  liquid. 
Resemblance  to  soluble  starch. 

2. — To  a  portion  of  the  solution  just  obtained  add  a  few  drops  of 
iodine  solution  (in  potassium  iodide).  A  reddish-brown  color  forms. 
Then  heat  the  contents  of  the  tube.  The  color  disappears,  to  reap- 
pear on  cooling.  Resemblance  to  erythrodextrin  and  to  starch-iodide. 
The  presence  of  pepton  interferes. 

3. — Boil  another  portion  of  the  glycogen  solution  with  Fehling's 
solution.     Note  the  result. 

4. — To  some  of  the  glycogen  solution  add  a  few  drops  of  HC1  and 
boil  a  few  minutes.  Glycogen-dextrin,  dextrose.  Then  cool  and  neu- 
tralize, and  test  a  portion  with  iodine;  another  portion  with  Fehling's 
solution.     Conpare  with  Exp.  2  and  3. 

5. — To  some  of  the  glycogen  solution  add  about  1  c.c.  of  saliva 
and  mix.  At  the  end  of  10  minutes  examine  a  portion  with  iodine; 
another  portion  with  Fehling's  solution.    What  is  the  result? 

Cellulose,  (C6H10O5)n . 

Cellulose,  or  wood-fiber,  is  present  in  all  higher  plants 
and  as  a  rule  in  the  lower  plants,  including  fungi  and  bac- 
teria. It  largely  makes  up  the  walls  of  the  cell.  Cellu- 
lose is  probably  formed  by  the  protoplasm  of  the  cell  out 
of  the  carbohydrates  that  result  from  the  assimilation  of 
the  carbonic  acid  of  the  air.  The  molecule  of  cellulose  is 
probably  much  more  complex  than  that  of  starch.  More- 
over, it  is  probable  that  there  are  various  distinct  cellulose 
bodies.     Tunicin  or  animal  cellulose  is  found  in  some  lower 


CARBOHYDRATES.  33 

animals,  as  the  tunicata,  and  is  identical  with  plant  cellu- 
ose  and    yields  on  decomposition  dextrose.     Cellulose  has 
been  reported  in  the  lungs,  blood  and  pus  of  tuberculous 
patients  (Freund). 

Cellulose  is  characterized  by  its  difficult  solubility.  It 
is  insoluble  in  water,  alcohol,  dilute  acids  or  alkalis.  It  is 
soluble  in  an  ammoniacal  solution  of  copper  oxide  or 
Schweizer's  reagent,  and  from  this  solution  it  can  be  pre- 
cipitated, unaltered,  in  an  amorphous  form  by  acids,  alco- 
hol or  water.  Cellulose  is  furthermore  characterized  by 
its  reaction  with  iodine  and  concentrated  sulphuric  acid. 
Treated  with  concentrated  sulphuric  acid  and  with  iodine 
it  gives  a  blue  color.  This  is  due  to  a  so-called  amyloid 
substance  which,  however,  is  not  identical  with  the  amy* 
loid  found  in  the  animal  body.  Indeed  the  latter  is  not  a 
carbohydrate  but  probably  a  proteid.  In  place  of  H2S04, 
zinc  chloride  can  be  used. 

It  does  not  melt  on  heating  but  turns  brown  and  event- 
ually decomposes,  yielding  various  products,  some  of  which 
have  considerable  commercial  importance.  Thus,  there  is 
formed  methyl  alcohol  (wood-spirit),  acetic  acid  (wood- 
vinegar),  and  creosote  (wood-tar). 

Concentrated  sulphuric  acid  dissolves  cellulose,  and  if 
this  solution  is  treated  at  once  with  water  a  gelatinous 
precipitate  of  soluble  cellulose  or  amyloid  forms.  If  the 
acid  is  allowed  to  act  longer,  or  the  solution  is  heated,  no 
precipitation  takes  place  on  dilution.  When  paper  is  rap- 
idly immersed  in  concentrated  sulphuric  acid  to  which  % 
its  volume  of  water  has  been  added,  and  is  then  washed  in 
water,  amyloid  which  is  first  formed  is  precipitated  on  the 
paper.     The  result  is  the  tough  parchment  paper. 

When  the  solution  of  cellulose  in  sulphuric  acid  is 
allowed  to  stand  for  some  time,  then  diluted  with  water 
and  boiled,  glucose  forms.  Some  kinds  of  cellulose  yield 
mannose.  Unlike  starch,  boiling  with  dilute  H2S04  has  but 
little  effect. 


34  PHYSIOLOGICAL  CHEMISTRY. 

With  concentrated  nitric  acid,  or  a  mixture  of  nitric 
and  sulphuric  (1—3)  acids,  it  forms  various  so-called  nitro- 
celluloses.  These  compounds  are  made  use  of  in  several 
important  preparations.  Thus,  collodium,  which  is  used  in 
surgery  and  in  photography,  is  a  mixture  of  tri-  and  tetra- 
nitro-cellulose  dissolved  in  ether.  Gun-cotton  or  pyroxylin 
is  a  mixture  of  the  tetra-  and  hexa-nitrates.  Smokeless 
powder,  which  has  revolutionized  modern  warfare,  may  be 
pure  gun-cotton,  or  gun-cotton  mixed  with  nitrate  of  barium 
and  potassium,  or  gun-cotton  mixed  with  nitro-glycerin  in 
different  proportions  (nobelite,  cordite,  explosive  gelatin). 
Powders  are  also  made  out  of  nitro-phenol  (picric  acid)  and 
out  of  nitro-naphthalens. 

A  mixture  of  nitro-cellulose  and  cellulose  can  be  drawn 
out  into  long  glistening  threads  resembling  silk  (wood- 
silk).  The  cellulose  which  is  the  basis  of  ordinary  paper  is 
obtained  from  wood  by  heating  it  with  calcium  sulphite 
under  pressure. 

Cellulose  has  been  obtained  in  the  shape  of  sphaero 
crystals  or  minute  needles.  Cotton  and  linen  threads  and 
Swedish  filter  paper  are  practically  pure  cellulose.  In  the 
dry  condition  it  is  permanent  but  in  the  presence  of  water 
it  readily  undergoes,  under  the  influence  of  bacteria,  fer 
mentative  decomposition  giving  rise  to  marsh  gas.  This 
bacterial  decomposition  takes  place  in  the  intestines  and 
marsh  gas,  acetic  and  butyric  acids  are  formed.  The  cellu- 
lose of  the  food  increases  the  peristaltic  action  of  the  intes- 
tines and  consequently  considerable  nitrogen  may  escape 
absorption. 

1. — Examine  under  the  microscope  and  sketch,  cotton,  linen,  silk 
and  wool  fibers,  also  hair.  The  linen  fibers  are  a  hollow  tube  with  a 
thick  wall  and  hence  retain  their  shape,  whereas  the  cotton  fibers 
have  thin  walls  which  readily  collapse  and  produce  the  twisted  char- 
acter. 

2.— Tear  up  a  little  "washed"  filter  paper  into  small  shreds  (or 
use  cotton)  and  warm  with  fresh  Schweizer's  reagent.     The  cellulose 


CARBOHYDRATES.  35 

dissolves.  Acidulate  the  solution  with  acetic  acid  when  it  precipi- 
tates in  an  amorphous  form.  Schweizer's  reagent  is  obtained  by 
adding  sodium  hydrate  to  a  solution  of  copper  sulphate  in  the  presence 
of  NH4C1.  The  copper  hydrate  precipitate  is  filtered  off,  washed  and 
dissolved  in  20%  ammonium  hydrate. 

3. — Immerse  some  shreds  of  "washed"  filter  paper,  or  cotton,  in 
a  strong  solution  of  potassium  hydrate  (1 — 1).  Allow  the  reagent  to 
act  for  10 — 15  minutes  till  the  paper  becomes  gummy.  Then  transfer 
to  a  dish  of  water,  and  wash  thoroughly,  then  acidulate  with  a  little 
dilute  hydrochloric  acid  and  add  some  iodine  solution.  A  blue  color, 
due  to  amyloid,  results. 

4. — To  some  cotton  or  shreds  of  paper  add  5 — 10  c.c.  of  cold  sul- 
phuric acid.  As  soon  as  solution  results  take  a  portion  of  it,  cool  and 
dilute  with  water.  A  gummy  precipitate  of  amyloid  forms.  Add 
iodine  solution,  it  colors  blue. 

Allow  the  remainder  of  the  acid  solution  to  then  stand  for  some 
time,  then  dilute  with  water  and  boil  for  J^hour;  cool,  neutralize  with 
potassium  hydrate  and  test  with  Fehling's  solution  for  sugar. 

5. — Dilute  some  sulphuric  acid  with  one-half  its  volume  of  water, 
and  cool  the  mixture.  Then  immerse,  for  a  few  seconds,  an  ordinary 
filter  paper;  remove  at  once  and  wash  in  tap-water.  The  tough 
parchment-paper  results. 

The  Mitscherlich   Polarimeter. 

The  instrument  consists  of  two  Nicol  prisms,  the  polarizer  and 
analyzer,  enclosed  in  brass  tubes  and  supported  in  such  a  way  that 
they  can  be  rotated;  the  tube  containing  the  analyzer  has  a  pointer 
attached  which  measures  the  amount  of  its  rotation  upon  a  circle 
graduated  in  degrees.  Between  the  two,  Nicols  is  placed  the  observa- 
tion tube,  a  brass  tube  exactly  200  m.m.  long,  the  ends  of  which  are 
closed  with  glass  plates;  this  holds  the  solution  to  be  tested. 

Adjust  the  instrument  as  follows: 

Place  a  lamp  behind  the  polarizer  and  fill  the  observation  tube 
with  distilled  water;  set  the  pointer  at  0°,  and  then  rotate  the  polar- 
izer until  the  field  becomes  darkest.  The  polarizer  must  not  be 
moved  again. 

As  the  instrument  now  stands,  the  two  Nicols  have  their  section 
at  right  angles.  If  the  analyzer  is  rotated,  one  way  or  the  other,  the 
field  gradually  becomes  brighter  and  is  brightest  when  the  pointer  is 


36  PHYSIOLOGICAL  CHEMISTRY. 

at  90°,  the  sections  of  the  prisms  now  being  parallel.     The  field  will 
be  dark  again  at  180°. 

Starting  with  the  instrument  adjusted  as  above,  fill  the  observa- 
tion tube  with  the  solution  to  be  tested.  If  this  has  the  power  of 
rotating  the  plans  of  polarization,  the  field  appears  bright.  The  ana- 
lyzer must  now  be  turned  to  the  right  or  left  till  the  field  again 
becomes  darkest,  thus  compensating  for  the  rotatory  power  of  the 
solution.  This  shows  whether  the  substance  is  dextro  or  lasvo-rota- 
tory.  By  knowing  the  length  of  the  tube,  the  concentration  of  the 
solution,  and  the  number  of  degrees  through  which  the  analyzer  was 
turned,  the  Specific  Rotatory  Power  can  be  calculated. 

The  Soleil-Ventzke  Saccharimeter. 

This  instrument  is  used  only  for  the  purpose  of  determining  the 
percentage  of  cane  sugar  in  a  given  sample. 

It  consists  of  two  Nicol  prisms,  the  analyzer  and  polarizer,  and 
the  observation  tube  placed  between  them.  Between  the  polarizer 
and  source  of  light  is  the  regulator,  a  Nicol  prism  and  a  quartz  plate, 
for  the  purpose  of  changing  the  colors.  Between  the  analyzer  and 
tube  is  the  compensator:  this  consists  of  two  wedge-shaped  plates, 
one  fixed,  and  the  other  capable  of  being  slid  over  it,  thus  increasing 
or  diminishing  the  thickness  of  the  crystal  through  which  the  polar- 
ized ray  passes.  Fastened  to  the  movable  plate  is  a  scale  graduated 
so  that  it  can  be  read  to  tenths  of  one  per  cent;  the  reading  is  done 
by  means  of  a  vernier  and  telescope.  The  source  of  light  is  a  lamp 
placed  back  of  the  polarizer. 

With  the  scale  reading  at  0°,  and  the  tube  filled  with  distilled 
water,  the  field  appears  as  a  colored  circle  divided  vertically,  and 
both  halves  of  exactly  the  same  shade  of  color.  This  color  may  be 
changed  by  simply  rotating  the  regulator.  For  most  persons  the 
"  sensitive  tint"  is  a  rose  violet. 

Now  fill  the  tube  with  a  solution  of  cane  sugar,  prepared  as  given 
below.  The  plane  of  polarization  is  deviated,  and  the  two  disks  are 
of  different  colors.  Then  turn  the  screw  of  the  compensator  till  the 
disks  are  again  of  the  same  shade,  thus  compensating  for  the  devia- 
ting effect  of  the  sugar.  The  percentage  of  cane  sugar  can  now  be 
read  distinctly  from  the  scale. 

The  instrument  is  so  made  that  with  a  solution  of  pure  cane  sugar 
containing  26,048  grms.  in  100  c.c.  at  17.5°  c.  the  reading  will  be  100  %. 
Consequently,  in  making  a  determination,  dissolve  26,048  grms.  of  the 
substance  in  distilled  water  at  17.5°,  and  dilute  to  100  c.c.  The  read- 
ing obtained  will  be  the  percentage  of  cane  sugar  in  the  substance. 


CHAPTER    III 
PROTEINS. 

Representatives  of  this  group  are  found  in  every  living- 
organism,  animal  or  vegetable.  They  are  present  within 
the  cell  as  an  essential,  integral  part  of  protoplasm,  and 
are  likewise  always  present  in  the  fluids  without  the  cell. 
In  addition  to  C,  H,  and  O  they  contain  N  and  S,  and  some 
have  P  and  even  Pe.  In  the  plant  these  substances  are  made 
from  the  inorganic  compounds,  ammonia,  nitrates,  nitrites, 
sulphates,  etc.,  whereas  in  herbivorous  animals  they  are 
derived  from  the  vegetable  food  in  which,  during  life,  these 
bodies  have  been  elaborated.  The  carnivorous,  or  omni- 
vorous animal  in  turn  builds  up  these  products  from  those 
contained  in  the  meat,  or  mixed  diet  respectively.  The 
animal  organism  cannot  make  protoplasm,  hence  live  and 
grow,  on  inorganic  nitrogen,  sulphur  and  phosphorus. 
These  elements  are  supplied  only  through  the  proteins 
existing  ready  made  in  our  food.  However,  not  all  the 
members  of  this  group  are  capable  of  sustaining  life;  this 
is  notably  true  of  the  albuminoids.  Those  members  are  of 
utility  as  real  food  which,  when  acted  upon  by  the  digestive 
fluids,  yield  peptons  which  in  turn  can  be  reconstructed  into 
serum  albumin  and  globulin. 

The  members  of  this  group  constitute  by  far  the  most 
complex  bodies  known  to  the  chemist.  While  their  percent- 
age  composition  can  often  be  determined  quite  readily  it  is 
otherwise  with  their  molecular  composition.  Undoubtedly 
thousands  of  atoms  may  be  contained  in  one  molecule.  On 
complete  cleavage  with  acids  they  yield  final  products  as 
ammonia;  organic  bases  as  lysin,  histidin,  arginin;  and 
amido  acids  as  leucin,  tyrosin,  etc. 

It  may  be  said  in  this  connection  that  considerable  con- 


38  PHYSIOLOGICAL   CHEMISTRY. 

fusion  exists  regarding  the  usage  of  certain  terms.  In 
English  works  the  term  proteid  is  used;  first,  as  a  general 
designation  for  the  entire  group,  i.  e.,  in  place  of  protein; 
second,  to  denote  one  of  the  subgroups,  namely,  the  albu- 
minous bodies.  On  the  other  hand  German  writers  use  it 
to  designate  the  more  complex  albuminous  bodies. 

The  following  table  is  essentially  that  of  Wroblewski. 

CLASSIFICATION   OF   PROTEINS. 

I.  Albuminous  Bodies;  these  contain  C,  H,  N,  O,  S; 
some  have  P. 

1. — Albumins  =  serum  albumin,  egg  aibumin^lactalbumin, 
muscle  albumin,  plant  albumin,  etc. 

2.— Globulins  =  serum  globulin,  egg  globulin,  lactoglobulin, 
fibrinogen,  myosin,  plant  globulins,  vitellin  (?)  etc. 

3. — Soluble  in  alcohol  —  chiefly  found  in  plants. 

4. — Albuminate. 

5. — Acid  albumin  =  syntonin,  etc. 

6. — Coagulated  albuminous  bodies  =  fibrin,  paracasein, 
heat  coagulated  albumins,  etc. 

II.  Proteids,  or  complex  albuminous  bodies,  which 
on  cleavage  yield  members  of  the  preceding  group. 

1. — Glycoproteids  =  mucin,  mucoids. 

2. — Haemoglobins. 

3. — Nucleo-albumins. 

4. — Caseins. 

5. — Nucleins. 

6. — Amyloid. 

7.— Histon  (?). 

III.  Albuminoids,  or  albumin — like  bodies. 

a.  Skeletal  or  support  substances. 

1. — Keratins. 

2. — Elastins. 

3.— Collagens  =  collagen,  gelatin,  etc. 

b.  Albumoses  and  peptons. 

c.  Enzymes. 

1. — Proteolytic  =  pepsin,  trypsin,  papayotin,  etc. 

2. — Amylolytic  =  diastase,  invertin,  etc. 

3. — Pat-splitting  =  steapsin. 

4. — Glucoside-splitting. 

5. — Amid-splitting  =  urase,  etc. 

6. — Coagulating  —  rennet. 


PROTEINS.  39 

I.     Egg   Albumin. 

Apply  the  following-  tests  which  are,  more  or  less,  general  reac- 
tions for  proteins  to  a  2%  solution,  unless  otherwise  indicated,  of  egg 
albumin.  A  white  of  an  egg  is  carefully  poured  into  an  evaporating 
dish,  then  cut  up  with  scissors,  and  20  c.c.  of  the  liquid  is  diluted  to 
1  liter  (1  —  50  =  2  % ).  After  thorough  shaking  in  a  cylinder  the  liquid  is 
filtered  and  the  clear  filtrate  employed  for  the  tests.  Observe  the 
frothing  of  the  liquid  on  shaking. 

Dilute  another  portion  of  2  c.c.  of  the  egg  albumin  to  10  c.c.  and 
shake  thoroughly  (1 — 5,  =  20%);  also,  dilute  2  c.c.  to  20  c.c.  and  shake 
till  thoroughly  mixed  (1—10,  =  10%). 

COLOR  REACTIONS  OF  PROTEIDS. 

The  following-  color  tests  (1 — 6)  are  general  reactions  for 
proteids: 

1. — Biuret  test. — To  the  albumin  solution  (1 — 50)  add  an  equal  vol- 
ume of  strong  sodium  or  potassium  hydrate.  Then  heat  to  boiling 
and  add  1 — 2  drops  of  very  dilute  CuS04  solution.  The  solution  becomes 
colored,  pink  to  violet,  according  to  the  amount  of  copper  sulphate 
used.  An  excess  of  copper  must  be  avoided.  Salts  of  nickel  give  a 
similar  reaction. 

Repeat  the  test  omitting  the  heat.     What  is  the  result? 

All  proteids  give  the  biuret  test,  some  more  readily 
than  others.  The  hydrated  proteids,  albumoses  and  pep- 
tons,  give  the  test  in  the  cold.  Gelatin  gives  in  the 
cold,  a  bluish  violet  color,  not  purple  red  as  in  the  case  of 
peptons. 

The  biuret  reaction  would  indicate  that  proteids  con- 
tain the  biuret  or  urea  group.  Diamids,  such  as  oxamid 
and  its  derivatives,  however,  give  similar  biuret  reactions, 
and  it  is  possible  that  such  diamid  groups  are  present  in 
the  proteid  molecule.  It  is  possible  to  remove  the  diamid 
group  and  the  modified  proteid  that  results  no  longer  gives 
the  biuret  reaction  (Schiff). 

2.  Mil hm's  reaction. — To  some  of  the  albumin  solution  (1 — 50)  add 
;i  few  drops  of  Millon's  reagent.     A  white  precipitate  forms  which  on 

4 


40  PHYSIOLOGICAL,  CHEMISTRY. 

boiling-  for  2 — 3  minutes  becomes  colored  red.    The  liquid  may  become 
likewise  red. 

This  reaction  is  due  to  the  aromatic  nucleus  contained 
in  the  proteid  molecule.     It  is  given  by  phenol,  tyrosin,  etc. 

Millon's  reagent  is  prepared  by  dissolving  in  the  cold 
one  part  of  mercury  in  one  part  by  weight  of  concentrated 
HN03  (1.40).  Gentle  heat  is  finally  applied  and  when  all  is 
dissolved  two  volumes  of  water  are  added.  The  mixture  is 
allowed  to  stand  for  some  hours  and  the  clear  liquid  is 
then  decanted  from  any  crystalline  sediment  that  may  be 
deposited. 

3. — Xanthoproteic  reaction. — To  some  of  the  albumin  solution  (1 — 50) 
add  an  equal  volume  of  concentrated  HN03.  Then  heat  to  boiling  till 
the  precipitate  turns  yellow  or  gives  a  yellow  solution.  Cool,  and  add 
an  excess  of  NH4OH  or  NaOH.  The  color  changes  to  an  orange 
yellow. 

This  test  can  be  always  incidentally  applied  to  the  precipitate 
or  liquid  obtained  in  Heller's  test,  or  in  the  nitric  acid  and  heat  test 
(I,  9  and  9a). 

4. — Adamkiewiez's  reaction. — To  2  c.c.  of  concentrated  H2S04  add 
about  4  c.c.  (two  volumes)  of  glacial  acetic  acid  and  mix.  To  the  mix- 
ture add  one  drop  of  the  undiluted  egg  albumin.  The  liquid  changes, 
slowly  on  standing,  more  rapidly  when  slightly  warmed  to  a  beauti- 
ful reddish  violet  color.  The  reaction  is  not  given  by  gelatin  or 
gelatin  pepton. 

The  presence  of  water  interferes  with  the  reaction.  It  is  there- 
fore desirable  to  use  the  dry  proteid  or  one  drop  of  a  concentrated 
solution.     The  spectrum  of  the  solution  resembles  that  of  urobilin. 

5. — Liebermann's  reaction. — To  about  3  c.c.  of  concentrated  HC1 
add  1 — 2  drops  of  undiluted  egg  albumin.  Boil  the  liquid  for  several 
minutes.  A  pink  to  a  violet  color  develops.  Too  much  water  inter- 
feres with  the  reaction. 

6. — Heat  some  albumin  with  concentrated  H2S04  and  a  little 
sugar.  A  red  color  results.  Excess  of  sugar  interferes  by  imparting 
a  dark  caramel  color  to  the  liquid. 

The  proteid  molecule  contains  one  or  more  aromatic 
groups.     This  is   seen  in  the  fact   that  on  decomposition 


PROTEINS.  41 

three  distinct  groups  of  aromatic  bodies  form.  Thus  we 
may  have  1st,  the  oxy-phenyl  group,  represented  in  phenol 
and  in  tyrosin;  2nd,  the  phenyl  group,  represented  in  phenyl- 
acetic  acid;  and  3rd,  the  indol  group  represented  by  indol 
and  skatol.  The  xanthoproteic  reaction  is  due  to  the  for- 
mation of  nitro-products  out  of  the  members  of  the  1st 
group.  Millon's  reaction  is  also  due  to  the  presence  of 
the  1st  group  of  compounds.  It  is  not  given  by  the  2nd  or 
3rd  groups.  The  Adamkiewicz  reaction  is  due  to  the  3rd 
group  of  products.  On  the  other  hand  the  Liebermann's 
reaction  is  apparently  not  due  to  the  aromatic  group. 

PRECIPITATION    REACTIONS    OP   PROTEIDS. 

7. — Take  four  test-tubes,  label  and  equip  as  follows:  To  tube  1  add 
1 — 2  c.c.  of  the  undiluted  egg  albumin;  to  tube  2  add  5  c.c.  of  the  egg 
albumin  solution,  1—5;  to  tube  3  add  5  c.c.  of  the  egg  albumin  solution, 
1 — 10:  and.  to  tube  4  add  5  c.c.  of  the  solution,  1—50.  Immerse  the  four 
tubes  in  a  boiling-  water-bath  for  5—10  minutes,  after  which  examine 
and  note  the  results.  Test  the  reaction  of  tubes  2,  3,  4.  Tube  1 
coagulates  solid,  whereas  tubes  2,  3,  4,  are  more  or  less  opalescent  but 
far  from  coagulation.  Dilution  of  egg  albumin  with  water  renders  it 
non-coagulable  by  heat.    Compare  with  similar  test  with  blood-serum. 

8. — In  each  of  four  test-tubes  place  5  c.c.  of  the  egg  albumin  solu- 
tion 1—50).  To  tubes  1  and  2  add  respectively  1  c.c.  and  0.2  c.c.  of  a  10% 
NaCl  solution.  To  the  tubes  3  and  4  add  respectively  one  and  five  drops 
of  a  1  %  acetic  acid  solution  l\  c.c.  of  a  glacial  acetic  diluted  to  100  c.c. ) 
To  a  fifth  tube  containing  5  c.c.  of  egg  albumin  solution  (1 — 10)  add  1  c.c. 
of  a  10%  NaCl  solution.  Immerse  the  five  tubes  in  a  boiling  water-bath 
for  about  five  minutes,  then  examine  and  note  the  results.  Test  the 
reactions  of  tubes  3  and  4.  In  experiment  7,  above,  tube  4,  which  can 
be  considered  as  a  control  for  this  experiment,  on  exposure  to  100° 
shows  only  a  very  slight  opalescence.  The  addition  of  a  small  amount 
of  NaCl  increases  the  opalescence  (tube  2);  the  same  amount  of  NaCl 
as  in  tube  1,  added  to  a  stronger  solution  of  albumin  (tube  5)  brings  on 
coagulation  on  heating;  and  a  larger  amount  brings  on  partial  coagu- 
lation on  the  walls  of  the  tube  (tube  1).  Now  add  one  or  two  drops  of  the 
1  %  acetic  acid  to  tubes  1,  2,  5,  and  to  tube  4  add  1  c.c.  of  10%  NaCl  and 
heat  again.     Prompt  and  complete  coagulation  results.    The  liquid  is 


42  PHYSIOLOGICAL  CHEMISTRY. 

clear.  In  tube  3  the  addition  of  one  drop  of  the  diluted  acid,  thus 
changing  the  liquid  to  a  very  slight  acid  reaction,  suffices  to  produce 
on  heating  a  precipitate.  A  very  slight  excess  of  the  acid  (as  in 
tube  4)  prevents  coagulation  by  heat.  If  NaCl,  however,  is  added 
coagulation  promptly  results. 

In  attempting  to  remove  albumin  completely  from  a  solution,  as 
in  the  case  of  urine,  it  should  be  remembered  that  very  dilute  solu- 
tions must  be  barely  acidulated  with  acetic  acid.  Furthermore,  that 
the  presence  of  NaCl  favors  coagulation  on  subsequent  heating. 

Albumin  coagulates  in  a  slightly  acid  or  neutral  solu- 
tion, especially  in  the  presence  of  a  neutral  salt  as  NaCl. 
Globulin  requires  a  neutral  salt  to  keep  it  in  solution  and 
this  moreover  favors  coagulation  on  heating".  Haemoglobin 
on  heating  decomposes  into  hasmatin  and  globulin;  the 
latter  as  just  stated  coagulates  on  heating  in  the  presence 
of  a  neutral  salt.  Nucleo-albumin  is  coagulated  or  thrown 
out  of  solution  by  acetic  acid  alone.  The  albumoses,  as  will 
be  seen  later,  are  precipitated  by  NaCl  and  the  precipitate 
unlike  albumin  and  globulin  dissolves  on  heating.  Peptons 
are  not  coagulated  by  heat. 

9. — To  about  5  c.c.  of  the  albumin  solution  (1 — 50)  add  an  equal  vol- 
ume of  concentrated  HN03  so  that  the  two  liquids  do  not  mix.  This 
is  done  by  allowing  the  acid  to  slowly  run  down  the  side  of  the  inclined 
tube.  A  white  cloud  forms  at  the  zone  of  contact  of  the  two  layers 
(Heller's  test).  Now  mix  the  two  liquids  and  gently  warm.  A  flocculent 
precipitate  separates.  Now  heat  the  mixture  to  boiling.  In  a  short 
time  the  precipitate  dissolves,  acid  albumin  forms,  and  the  liquid  is 
colored  yellow.  Cool  the  liquid  and  add  an  excess  of  NH^OH.  An 
orange  yellow  color  results  (Xanthoproteic  reaction). 

Egg  albumin  is  therefore  coagulated  by  HN03.  The  solution  of 
this  precipitate  on  boiling  shows  a  distinction  between  this  and  the 
serum  proteids. 

Mineral  acids,  such  as  HN03,  coagulate  albumin  and 
globulin.  The  albumoses  are  precipitated  by  HN03  espec- 
ially if  NaCl  is  present,  but  the  precipitate  readily  dis- 
solves on  the  application  of  heat  and  reappears  on  cooling. 
Peptons  are  not  precipitated  by  acids. 


PROTEINS.  43 

9«. — The  test  employed  most  often  for  the  detection  of  albumin 
(and  globulin)  in  the  filtered  urine  is  the  coagulation,  or  nitric  arid  and 
heat  test.  The  reaction  when  properly  carried  out  is  exceedingly  deli- 
cate. The  best  procedure  is  as  follows:  To  the  urine  add  some  con- 
centrated HN03  so  as  to  form  two  layers  (see  above).  A  precipitate 
or  cloud  indicates  albumin.  Now  mix  the  two  liquids  and  heat.  A 
persistent  flocculent  precipitate  is  due  to  albumin,  or  globulin,  or 
both.  Should  it  be  necessary  to  decide  whether  this  precipitate  is 
due  in  part  or  whole  to  albumin,  it  can  be  done  by  saturating  the 
urine  with  MgS04  according  to  directions  given  under  Globulin  Test  5. 

If  heat  is  applied  direct  to  the  urine  a  precipitate  of  phosphates 
may  form.  This,  however,  dissolves  readily  in  HNO,.  If  the  urine  is 
alkaline  the  HNOa  should  be  added  first  to  prevent  formation  of  alka- 
li-albuminate. 

Apply  the  test  as  just  given  to  some  albuminous  urine. 

10. — To  about  5  c.c.  of  the  albumin  solution  (1 — 50)  add  1 — 2  drops 
of  strong  acetic  acid,  then  add  1 — 2  drops  of  potassium  ferrocyanide. 
A  voluminous  precipitate  forms. 

This  is  a  very  delicate  test  for  proteins.  It  is  not 
given,  however,  by  peptons.  The  presence  of  NaCl  favors 
the  precipitation  of  the  albumoses.  Moreover  the  albumose 
precipitate  dissolves  on  heating  and  reappears  on  cooling. 

This  test  and  the  nitric  acid  heat  test,  given  above,  are 
commonly  employed  for  the  detection  of  albumin  in  the 
urine. 

If,  in  the  case  of  urine,  the  amount  of  the  precipitate  is 
small  and  its  nature  doubtful  it  should  be  transferred  to  a 
filter  and  washed.  The  precipitate  can  be  transferred  by 
means  of  a  glass  rod  to  a  test-tube  and  Millon's  reagent 
added.  If  on  heating  a  reddish  coloration  forms  it  indi- 
cates the  presence  of  a  proteid.  Another  procedure  is  to 
add  Yo  c.c.  of  the  boiling  Millon's  reagent  direct  to  the  preci- 
pitate on  the  filter  (Winternitz). 

11. — Strongly  acidulate  some  of  the  albumin  solution  (1 — 50)  with 
\\<  I.  then  add  a  few  drops  of  phosphotungstic  acid.  A  heavy  white 
precipitate  results.  It  is  given  by  all  proteids.  Phosphomolybdic 
acid  behaves  in  a  similar  manner. 


44  PHYSIOLOGICAL,  CHEMISTRY. 

12. — Acidulate  another  portion  as  above  and  add  a  few  drops  of  a 
solution  of  potassium  mercuric  iodide.  Note  the  results.  Why  was 
this  reagent  used  in  the  preparation  of  glycogen? 

13.— To  a  portion  of  the  albumin  solution  (1 — 50)  add  1 — 2  drops 
of  tannic  acid.  What  is  the  behavior  of  tannic  acid  to  starch?  To 
dextrin? 

14. — To  another  portion  of  the  solution  add  a  few  drops  of  picric 
acid.  A  yellow  voluminous  precipitate  forms.  This  reagent  is  used 
in  Esbach's  method  for  the  estimation  of  albumin  in  urine. 

The  reagents  employed  in  tests  10 — 14  inclusive  are 
sometimes  spoken  of  as  alkaloidal  regents  because  of  their 
reactions  with  the  vegetable  alkaloids  and  other  bases. 
They  are  general  reagents  for  proteids. 

15. — To  3  c.c.  of  the  albumin  solution  (1 — 50)  add  one  drop  of  mer- 
curic chloride.  A  heavy  white  cloud  or  precipitate  results.  Divide 
the  cloudy  liquid  into  two  portions. 

(a).  To  one  add  an  equal  volume  of  a  10%  solution  of  NaCl.  The 
precipitate  promptly  dissolves  even  if  mercury  is  in  large  excess. 

(&).  To  the  other  portion  add  two  volumes  of  the  undiluted  egg 
albumin  solution  and  mix.  The  precipitate  dissolves  if  too  much 
mercury  has  not  been  added. 

16. — To  another  small  portion  of  the  egg  albumin  solution  add 
1 — 2  drops  of  dilute  lead  acetate  and  note  the  result. 

17. — To  a  portion  of  the  solution  add  1 — 2  drops  of  silver  nitrate. 
A  voluminous  white  precipitate  forms  which  on  the  addition  of 
NH4OH  dissolves. 

Experiments  15,  16,  17,  are  made  with  the  salts  of  the 
heavy  metals  which  precipitate  most  of  the  proteids.  Why 
is  the  white  of  eggs  administered  in  case  of  poisoning  with 
corrosive  sublimate  or  with  salts  of  other  heavy  metals? 
Why  should  a  stomach  pump  be  subsequently  used? 

18.— To  about  3  c.c.  of  the  albumin  solution  (1—50)  add  10—15  c.c. 
of  strong  alcohol  and  mix.  If  no  precipitate  forms,  but  merely  a 
cloudiness,  then  add  %— l/2  c.c.  of  a  10%  solution  of  NaCl.  A  volumin- 
ous white  precipitate  results. 


PROTEINS.  45 

Alcohol  added  in  large  excess  (10  volumes  or  more)  pre- 
cipitates all  proteids.  The  presence  of  NaCl  favors  the 
precipitation. 

19. — Place  10  c.c.  of  egg  albumin  solution  (1 — 50)  in  a  small  beaker 
or  test-tube  on  foot.  Add  about  7  g.  of  powdered  (NH4)2S04  and 
immerse  in  a  water-bath  at  about  35°  for  half  an  hour.  Stir  fre- 
quently till  the  salt  ceases  to  dissolve.  Notice  the  heavy  white  pre- 
cipitate that  forms  (albumin  and  globulin).  When  saturated  transfer 
the  contents  to  a  dry  filter.     Test  the  filtrate: 

(a).     By  acetic  acid  and  heat. 

(b).     By  the  biuret  test,  in  the  cold. 

Are  the  results  positive  or  negative?     Explain. 

20. — Place  10  c.c.  of  the  egg  albumin  solution  (1 — 50)  in  a  small 
beaker,  as  above,  add  about  12  g.  of  MgS04  and  digest,  with  fre- 
quent stirring,  at  35°  for  about  half  an  hour.  Observe  that  only  a 
very  slight  cloud  or  precipitate  forms  (globulin).  Filter  through  a 
dry  filter  and  to  the  filtrate  apply  tests  a  and  b  as  in  Experiment  19. 
What  proteid  is  present  in  the  filtrate?  In  the  biuret  test  a  large 
excess  of  NaOH  should  be  added  owing  to  the  precipitate  of  Mg(OH)2 
that  forms. 

On  saturation  with  MgS04  globulin  is  precipitated 
whereas  albumin  remains  in  solution.  Saturation  with 
(NH4)2S04  throws  down  albumin,  globulin,  and  albumose, 
but  not  pepton. 

21. — Determination  of  the  coagulation  point  of  albumin. — Place  about 
5  c.c.  of  the  undiluted  egg  albumin  in  a  test-tube.  Close  the  tube 
with  a  stopper  through  which  passes  a  thermometer.  The  bulb  of 
the  thermometer  should  nearly  touch  the  bottom  of  the  tube  and 
should  be  completely  immersed  in  the  albumin.  Suspend  the  tube 
thus  equipped  in  a  large  beaker  of  water.  Fully  two-thirds  of  the 
tube  should  be  immersed.  Heat  gradually  the  water  in  the  beaker 
and  stir  continually  by  means  of  a  glass  rod  bent  at  right  angles# 
Note  the  temperature  at  which  the  albumin  clouds.  The  albumin 
then  becomes  sticky;  does  not  flow  readily  when  inclined  and  finally 
becomes  solid.     Note  the  coagulating  point  of  egg  albumin. 


46 


PHYSIOLOGICAL   CHEMISTRY. 


22. — To  20  c.c.  of  the  2%  albumin  solution  add  2 — 3  drops  of 
concentrated  HC1  and  boil.  No  precipitate  forms  owing-  to  the  for- 
mation of  an  acid  albumin.     Cool  the  solution. 

(a).  To  a  portion  add  an  excess  of  concentrated  HC1,  a  precipi- 
tate forms  that  is  difficulty  soluble  in  excess. 

(ft).  In  the  remainder  of  the  solution  place  a  litmus  paper  and 
add,  drop  by  drop,  very  dilute  NaOH.  Mix  the  contents  well  after 
each  addition  of  alkali.  As  soon  as  a  precipitate  or  cloud  forms  note 
the  reaction  of  the  liquid.  The  precipitate  of  albuminate  forms 
while  the  liquid  is  still  acid.  After  the  precipitate  has  formed  add 
2 — 3  drops  more  of  the  dilute  NaOH.  It  dissolves  at  once  to  form 
an  alkali  albuminate. 

23. — To  10  c.c.  of  the  albumin  solution  add  1 — 2  drops  of  NaOH 
solution  and  warm  gently  for  a  few  minutes.  An  alkali  albuminate 
forms.  Raise  the  solution  to  boiling.  It  does  not  coagulate.  Cool, 
add  litmus  paper  and  carefully  neutralize,  as  above,  with  dilute  HC1. 
What  is  the  result?    What  is  the  effect  of  a  slight  excess  of  HO? 

Report  the  results  obtained  with  egg  albumin  and  with 
the  proteids  subsequently  to  be  studied  in  a  tabular  form 
such  as  the  following-: 


Biuret  1 

Millon2 

Xanthoproteic  3 

Adamkiewicz  4 . 

Boiling 

Nitric  acid  9  

Acetic  and  ferrocyanide  10. 

Phosphotungstic  acid  11 

Pot.  mercuric  iodide  12..  

Tannic  acid  13  

Picric  acid  14 

Mercuric  chloride  15 

Lead  acetate  16 

Silver  nitrate  17 

Alcohol  18 

Ammonium  sulphate  19 

Magnesium  sulphate  20 


2 


§•§ 


GO 


2  ^ 


Ph 


PROTEINS.  47 

II.     Serum  Albumin  and   Serum  Globulin. 

Globulin. — It  is  usually  associated  with  albumin,  though 
it  may  sometimes,  as  in  the  urine,  occur  alone.  The  tests 
given  for  albumin,  as  well  as  the  general  proteid  reactions, 
are  also  given  by  globulin.  For  the  separate  recognition 
of  albumin  and  globulin,  when  both  are  present  in  solution, 
it  is  necessary  to  resort  to  precipitation  by  either  of  the 
following  methods: 

1. — Precipitation  with  MgSOt. — To  lOc.c.  of  blood-serum,  in  a  small 
beaker  or  test-tube  on  foot,  add  10  c.c.  of  saturated  MgS04  and  15  g. 
of  powdered  MgS04.  Immerse  the  beaker  or  tube  in  a  water-bath  at 
a  temperature  of  30 — 35°.  Stir  frequently  for  % — 1  hour,  until  the 
MgS04  ceases  to  dissolve  and  the  liquid  is  saturated.  The  globulin  is 
thrown  out  of  solution.  Transfer  the  liquid  and  precipitate  to  a 
small  filter.  Save  the  nitrate  (a)  which  contains  albumin.  When  the 
liquid  has  drained  through  wash  the  residue  2 — 3  times  with  saturated 
MgS04.  Finally  spread  out  the  filter  on  a  flat  surface,  transfer  the 
precipitate  by  means  of  a  spatula  to  about  20  c.c.  of  water.  Globulin 
when  pure  does  not  dissolve  in  water,  but  in  this  case,  owing  to  the 
presence  of  salts,  it  dissolves. 

Filter  the  solution  and  the  clear  filtrate  (b)  containing  the  globu- 
lin is  reserved  for  experiment  3. 

Apply  to  the  original  filtrate  (a)  which  contains  serum  albumin 
the  tests  enumerated  in  the  table.  Note  the  results.  Wherein  does 
egg  albumin  differ  from  serum  albumin?  Boil  a  portion  of  the  serum 
albumin  solution  to  coagulate  the  albumin.  Filter  and  apply  the 
biuret  test  to  the  nitrate.     What  is  the  result? 

2. — Precipitation  by  semi-saturation  with  (NHi)2SOi. — To  10  c.c.  of 
blood  serum  as  above,  add  10  c.c.  of  saturated  (NH4)2S04.  Immerse  in 
a  water-bath  at  30—35°  for  about  yi  hour  and  stir  frequently.  Then 
transfer  the  contents  to  a  small  filter.  Save  the  filtrate  (A)  which 
contains  albumin.  Wash  the  residue  on  the  filter  2—3  times  with 
semi-saturated  (NH4)2SO«.  Finally  spread  out  the  filter  on  a  flat  sur- 
face, transfer  the  precipitate  to  about  20  c.c.  of  water.  The  globulin 
precipitate  dissolves  for  the  reasons  given  above  under  1. 

Filter  the  solution  and  combine  the  clear  filtrate  (B)  with  the 
corresponding  filtrate  from  experiment  1.  The  resulting  solution  is 
used  for  experiment  3. 


48  PHYSIOLOGICAL  CHEMISTRY. 

The  precipitate  obtained  by  this  method  is  larger  than  that 
obtained  by  the  MgS04  method.     The  liquid  filters  much  easier. 

3. — Separation  of  salts  from  globulin  by  dialysis. — Place  the  com- 
bined filtrates  (&  and  B)  in  a  dialyzer  and  dialyze  against  running 
water.  Every  day  remove  a  few  drops  of  the  liquid  from  the  dialyzer 
with  a  pipette  and  add  this  to  some  dilute  BaCl2  solution.  The  dia- 
lysis should  continue  till  the  sulphates  are  completely  removed.  This 
may  require  3 — 5  days.  In  warm  weather  to  prevent  decomposition  it 
is  well  to  add  a  few  drops  of  thymol. 

When  the  sulphates  have  dialyzed  out  the  globulin  is  thrown  out 
of  solution  as  a  white  granular  precipitate.  Now  transfer  the  con- 
tents of  the  dialyzer  to  a  small  beaker.  Pour  into  the  dialyzer  about 
20  c.c.  of  a  2%  solution  of  NaCl  and  gently  agitate  to  dissolve  any  pre- 
cipitated globulin.  Add  this  saline  solution  to  the  contents  of  the 
beaker  and  stir  till  the  globulin  dissolves.  Finally  allow  the  liquid  to 
stand  for  a  while,  then  filter.  The  clear  filtrate  now  contains  pure 
globulin.     Observe  the  frothing  of  the  liquid  on  shaking. 

To  this  solution  of  globulin  apply  the  tests  enumerated  in  the 
table  on  page  46.  Make  careful  records  of  the  results  obtained.  The 
presence  of  NaCl  will  interfere  with  tests  16  and  17. 

Boil  a  portion  of  the  globulin  solution  to  coagulate  the  globulin.. 
To  the  filtrate  apply  the  biuret  test. 

4. — To  10  c.c.  of  blood-serum  add  an  equal  volume  of  saturated 
(NH4)2S04.  Then  add  8  g.  of  powdered  (NH4)2S04  and  immerse  in  a 
water-bath  at  30 — 35°,  stirring  frequently,  for  about  y2  hour.  The 
liquid  becomes  saturated  with  (NH4)2S04  and  a  precipitate  forms. 
Finally  transfer  to  a  filter.  Notice  the  perfect  clearness  of  the 
filtrate. 

Test  a  portion  of  the  filtrate  by  boiling;  another  portion  with 
tannic  acid. 

To  another  portion  apply  the  biuret  test  in  the  cold. 

The  absence  of  proteids  in  the  filtrate  demonstrates  that  albu- 
min and  globulin  are  completely  precipitated  on  saturation  with 
(NH4)2S04.  The  absence  of  a  biuret  reaction  indicates  the  absence  of 
a  pepton. 

5*. — Detection  of  globulin  in  the  urine. — As  indicated  under  albumin 
(Experiment  9a,  page  43),  the  ordinary  tests  for  albumin  are  also^ 
given  by  globulin.     In  order  to  ascertain  positively  which  of  the  two, 

*  Experiments  or  methods  designated  by  an  asterisk  are  omitted  in  the  ordinary- 
laboratory  course. 


PROTEINS.  49 

or  if  both  are  present,  it  is  necessary  to  resort  to  the  saturation 
method  with  MgS04.  For  this  purpose  100  c.c.  of  the  urine  can  be 
taken  and  neutralized.  120  g".  of  powdered  MgS04  are  then  added  and 
the  liquid  kept  at  30 — 35",  with  frequent  stirring,  till  the  MgS04 
ceases  to  dissolve.  If  globulin  is  present  a  precipitate  will  form. 
This  can  be  removed  by  nitration,  washed  with  saturated  MgS04  solu- 
tion, and  finally  dissolved  in  water  (See  page  47,  Globulin  Exp.  1). 
This  solution  of  the  MgS04  precipitate  can  now  be  tested.  It  coagu- 
lates on  heating  especially  if  slightly  acidified  with  acetic  acid.  It 
gives  the  nitric  acid  and  heat  tests,  also  the  biuret  reaction. 

The  filtrate  from  the  MgS04  precipitate  contains  albumin  if  any 
is  present.  The  tests  just  given  applied  to  this  filtrate,  if  positive, 
prove  the  presence  of  albumin. 


III.     Albumose. 

This  compound,  or  rather  group  of  compounds,  can  be 
readily  prepared  from  Witte's  or  Schuchardt's  commercial 
pepton  since  this  consists  largely  of  albumoses.  Albumoses 
are  precipitated  by  saturation  with  (NH4)2S04  or  with  NaCl 
in  an  acid  solution. 

A  20%  solution  of  the  commercial  pepton  is  used.  The 
powder  readily  dissolves  especially  if  the  liquid  is  warmed 
and  thoroughly  stirred. 

1. — Place  seme  of  the  solution  in  a  test-tube  and  heat  to  boiling. 
The  liquid  does  not  coagulate,  thus  indicating  the  absence  of  albumin 
and  globulin. 

2.— To  10  c.c.  of  the  solution  add  10  c.c.  of  saturated  (NH4)„S04 
solution  and  about  8  g.  of  powdered  (NH4)2S04.  Saturate  the  liquid  in 
a  water-bath  at  about  30 — 35°  according  to  the  directions  given  in 
experiment  4.  page  48.  Notice  the  sticky  precipitate  that  adheres 
to  the  rod  and  to  the  sides  of  the  beaker  or  tube.  Since  albumin  and 
globulin  are  absent,  as  ascertained  from  preceding  experiment,  the 
precipitate  that  forms  consists  of  albumoses.  Transfer  the  precipi- 
tate to  a  filter  and  wash  with  about  10  c.c.  of  saturated  (NH4)S04 
solution. 

Save  the  filtrate  (A)  for  subsequent  tests  for  pepton. 


50  PHYSIOLOGICAL  CHEMISTRY. 

By  means  of  a  glass  rod  gather  the  sticky  albumose  precipitate 
and  transfer  it  to  about  20  c.c.  of  water  in  a  test-tube.  While  stirring, 
heat  the  liquid  carefully  and  the  albumose  dissolves  completely. 

With  this  aqueous  solution  of  pure  albumose  make  the  following 
tests,  employing  small  quantities  of  the  liquid,  about  1  c.c. 

(a).     Heat  a  portion  to  boiling.     It  does  not  coagulate. 

(&).  To  a  portion  add  HNOa,  drop  by  drop.  A  slight  precipitate 
may  form  which  dissolves  and  gives  a  yellow  solution.  If  there  is  no 
permanent  precipitate  add  some  saturated  NaCl,  drop  by  drop,  till  a 
precipitate  does  form.  Now  gently  heat  the  contents  of  the  tube. 
The  precipitate  dissolves  and  on  cooling  reappears.  This  reaction  is 
characteristic  of  albumoses. 

In  the  absence  of  NaCl  some  of  the  albumoses,  especially  deutero- 
albumose,  do  not  give  a  precipitate  with  HN03.  An  excess  of  NaCl, 
however,  should  be  avoided,  since  in  that  case  the  albumose  precipi- 
tate does  not  dissolve  completely  on  heating. 

(c).  To  a  portion  of  the  solution  add  a  few  drops  of  acetic  acid 
(1 — 10)  and  2 — 3  drops  of  potassium  ferrocyanide.  If  no  precipitate 
forms  add  NaCl  according  to  directions  given  above  under  (ft).  A  pre- 
cipitate will  then  form  and  on  heating  gently  it  dissolves.  On  cooling 
the  solution  it  reappears. 

This  reaction,  like  the  preceding,  is  also  characteristic  of  albu- 
moses.    A  certain  amount  of  NaCl  is  necessary  as  in  the  HN03  test. 

(d).  To  about  lc.c.  of  the  solution  add  1 — 2  drops  of  dilute  acetic 
acid  and  about  5  c.c.  of  a  saturated  NaCl  solution.  A  precipitate 
or  cloudiness  results.  On  heating  this  disappears,  to  reappear  on 
cooling. 

To  the  remainder  of  the  solution  of  albumose  apply  the  tests 
given  in  the  table  (page  46)  and  note  the  result.  In  which  of  these 
reactions  will  the  presence  of  chlorides  and  of  ammonium  salts 
interfere? 

Apply  the  biuret  test  without  the  aid  of  heat.  The  hydration 
proteids  give  this  reaction  readily  in  the  cold. 

3*. — Detection  of  albumose  in  the  urine. 

The  reactions  given  above  under  2,  especially  &,  c,  and 
d,  are  characteristic  of  albumoses.  To  detect  albumose  in 
the  urine,  or  in  other  liquids,  it  is  necessary  first  to  remove 
the  albumin  and  globulin.     This  can  be  readily  done  by 


PROTEINS.  51 

acidifying-  slightly  with  acetic  acid  and  applying  heat. 
The  albumin  and  globulin  coagulate.  To  the  filtrate  the 
biuret  test  can  be  applied.  If  the  result  is  negative  it  indi- 
cates the  absence  of  albumoses  and  also  of  pepton.  If, 
however,  the  result  is  positive,  it  is  due  either  to  albumoses 
or  to  pepton,  or  to  both.  The  tests  given  above  under  26, 
c,  and  d,  can  now  be  applied  and  if  positive,  the  presence  of 
albumose  is  demonstrated.  If  these  tests  fail  the  positive 
biuret  reaction  is  due  to  pepton. 

Another  method  for  the  detection  of  albumose  consists 
in  saturating  the  urine  with  salt,  acidulating  with  acetic 
acid  and  boiling.  The  albumin  and  globulin  coagulate;  the 
albumose  is  in  solution.  Filter  boiling  hot.  If  the  filtrate 
on  cooling  gives  a  precipitate  it  is  due  to  albumose.  To 
the  filtrate  apply  the  biuret  reaction. 

Compare  methods  of  detection  given  under  pepton. 


IV.     Pepton. 

Pepton  is  not  precipitated  by  (NH4)2S04.  The  nitrate  (A)  ob- 
tained in  experiment  2  under  albumose  therefore  contains  pepton  if 
it  be  present. 

To  this  filtrate  apply  the  biuret  test  in  the  cold.  A  positive 
reaction  is  due  to  pepton.  As  previously  indicated  the  hydrated  pro- 
teids,  as  a  rule,  do  not  require  heat  in  order  to  give  the  biuret 
reaction. 

To  obtain  a  pure  solution  of  the  pepton  it  would  be  necessary  to 
resort  to  dialysis,  or  to  treatment  with  baryta  on  a  water-bath  to 
remove  the  iNH4)2S04. 

To  the  original  filtrate  containing-  pepton  apply  the  tests  given 
in  the  table  on  page  46  and  note  the  results.  With  which  of  these 
reactions  will  the  (NH4)2S04  present  interfere? 

*  Detection  of  pepton  (Hofmeister). 

To  about  500  c.c.  of  the  urine,  or  to  an  aqueous  extract 
of  the  tissue  to  be  examined,  made  at  about  40°,  add  just 
enough  lead  acetate  to  give  a  strong  precipitate  and  filter. 


52  PHYSIOLOGICAL  CHEMISTRY. 

This  removes  mucin.  Test  the  nitrate  for  albumin  and  if 
present  remove  in  the  following-  manner:  Add  a  little 
sodium  acetate  and  then  concentrated  ferric  chloride  till 
the  mixture  is  blood  red  in  color.  Then  neutralize  with 
potassium  hydrate  (or  leave  slightly  acid),  boil,  cool  and 
filter.  The  filtrate  should  give  no  precipitate  with  acetic 
acid  and  potassium  ferrocyanide  (absence  of  iron  and  of 
albumin).  If  it  is  perfectly  free  from  albumin  make  the 
following  tests: 

1. — Add  acetic  acid  and  phosphotungstic  acid — a  cloud- 
iness forms  on  standing  if  pepton  is  present. 

2. — If  pepton  is  indicated  by  the  above  trial  it  can  be 
isolated  by  the  following  method:  Add  0.1  volume  of  con- 
centrated hydrochloric  acid  and  then  phosphotungstic  acid, 
also  acidulated  with  hydrochloric  acid,  as  long  as  a  precipi- 
tate continues  to  form.  Filter  at  once  and  wash  with  dilute 
sulphuric  acid  (3  to  5  c.c.  in  100  c.c.  of  water),  till  the  filtrate 
is  colorless.  While  the  precipitate  is  still  moist  mix  it  with 
an  excess  of  powdered  barium  hydrate,  add  a  little  water, 
gently  warm  for  a  short  time  and  filter.  To  the  filtrate 
which  contains  pepton  apply  the  biuret  test. 

The  Hofmeister  method  strictly  speaking  does  not  indi- 
cate true  peptones,  but  rather  albumose. 

Another  method  for  the  detection  of  pepton  is  based 
upon  its  behavior  to  (NH+)2S04.  The  method  as  employed 
by  Devoto  is  as  follows;  To  200 — 300  c.c.  of  the  urine  add 
80%  by  weight  of  (NH4)2S04.  This  is  added  to  urine  even  if 
albumin  and  globulin  are  absent  in  order  to  remove  nucleo- 
albumin.  Warm  the  mixture  on  the  water-bath  till  the  salt 
dissolves.  This  will  occur  in  10 — 15  minutes.  Now  place 
the  beaker  in  a  steam  sterilizer  for  30 — 40  minutes  or  longer. 
The  albumin  coagulates  completely  irrespective  of  the  reac- 
tion of  the  fluid.  The  mixture  is  allowed  to  cool,  then  fil- 
tered. The  filtrate  can  be  tested  for  the  biuret  reaction.  If 
positive  pepton  is  present.     It  can  further  be  precipitated 


PROTEINS.  53 

with  tannic  acid.  Under  these  conditions  deutero-albumose 
is  not  completely  precipitated  and  hence  can  be  easily  mis- 
taken for  true  pepton. 

The  residue  on  the  filter  can  be  washed  with  hot  water 
till  the  filtrate  ceases  to  give  a  test  for  BaS04.  If  the  filter 
has  previously  been  dried  and  weighed,  and  is  now  ag'ain 
dried  and  weighed  the  difference  is  due  to  the  albumin  and 
globulin.     (See  estimation  of  albumin  and  globulin). 

The  first  portions  of  the  hot  wash-water  are  collected , 
combined  and  tested  for  the  biuret  reaction.  If  positive  it 
is  ordinarily  said  to  be  due  to  pepton  (Devoto,  Jaksch),  but 
in  reality  it  is  due  to  the  albumoses  which  are  not  rendered 
insoluble,  like  albumin  and  globulin,  and  dissolve  on  treat- 
ment with  hot  water. 

The  Hofmeister  method  it  is  evident  will  often  give 
positive  results  where  Devoto's  method  fails. 

In  reality  the  reaction  in  both  cases  as  indicated  above 
is  due  to  albumoses.  The  true  pepton  which  would  be  pres- 
ent in  the  filtrate  from  the  cold  saturated  solution  seems  to 
be  very  rare,  if  at  all,  in  urine. 

As  used  in  a  clinical  way  the  term  "pepton"  includes 
pepton  and  albumoses.  Such  pepton  may  be  present, 
though  not  always,  in  the  blood  of  the  leukaemics  during 
life.  The  blood  obtained  from  deceased  leukaemics,  espec- 
ially if  decomposition  has  set  in,  is  rich  in  such  pepton. 
The  normal  liver  does  not  contain  pepton,  whereas  the 
spleen  does.  The  liver  and  spleen  of  leukaemics  are  rich  in 
such  pepton. 

A  better  process  for  the  detection  of  true  pepton  is  as 
follows: 

Saturate  the  solution  at  the  boiling-  point  with  ammon- 
ium sulphate  and  filter  while  boiling  hot.  Allow  the  filtrate 
to  cool,  decant  the  liquid  from  the  crystals  which  separate, 
dilute  strongly  and  precipitate  the  pepton  by  cautious  addi- 
tion of  tannic  acid.     Let  stand  for  24  hours,  then  filter. 


54  PHYSIOLOGICAL    CHEMISTRY. 

Boil  the  precipitate  for  a  few  minutes  with  baryta  water, 
filter,  and  from  the  filtrate  remove  the  excess  of  barium  by- 
passing- carbonic  acid.  Filter  off  the  barium  carbonate  and 
test  the  filtrate  for  biuret. 

V.     Gelatin. 


To  study  the  reactions  of  gelatin  a  2%  solution  of  the  best  French 
gelatin  (silver)  is  employed. 

1.—  Shake  up  some  of  the  solution.  Notice  the  foaming  of  the 
liquid. 

2. — To  a  portion  of  the  solution  add  some  bromine  water.  An 
abundant,  yellow,  sticky  precipitate  forms. 

3. — In  each  of  two  test-tubes  add  1 — 2  drops  of  saturated  HgCl2 
solution.  To  tube  1  add  about  5  c.c.  of.  the  gelatin  solution.  To  tube 
2  add  about  5  c.c.  of  water.  Then  add  to  each  tube  some  H2S 
water  and  heat.  Tube  1  is  dark  yellow,  but  contains  no  precipitate, 
whereas  tube  2  has  a  blackish  precipitate  of  HgS  and  the  liquid  is 
clear.  Gelatin  prevents  the  precipitation  of  many,  otherwise  insolu- 
ble, compounds.     Glycogen  possesses  a  similar  property. 

4. — To  the  gelatin  solution  apply  the  several  tests  given  in  the 
table  on  page  46.  Tabulate  the  results,  and  carefully  note  the  differ- 
ences. 

Observe  that  the  heavy  metals  do  not  precipitate  gelatin, 
whereas  the  other  proteids  are  precipitated.  Also,  that  gelatin  is 
not  precipitated  by  f  errocyanide  even  in  the  presence  of  NaCl,  and  in 
this  respect  it  resembles  pepton. 

The  xanthoproteic  reaction  is  weak  owing  to  the  absence  of  the 
phenol  group,  C6H5OH.  The  biuret  reaction  applied  to  the  cold  solu- 
tion of  gelatin  gives  a  bluish  violet  color,  whereas  pepton  gives  a 
purple  red.  Millon's  reagent  gives  a  white  precipitate  which  on  heat- 
ing becomes  red,  and  the  liquid  becomes  pink.  It  is  probable  that 
the  other  reactions  are  not  strictly  due  to  the  gelatin  but  to  an 
admixture  of  some  pepton  or  albumose. 

Whereas  albumin  contains  three  aromatic  groups  (see  page  40) 
gelatin  contains  but  one,  since  it  yields  phenyl  amidopropionic  acid 
or  tyrosin  on  decomposition. 


CHAPTER    IV. 

SALIVA. 

Saliva  is  a  mixture  of  the  secretions  of  the  parotid, 
submaxillary,  and  sub-lingual  glands.  The  reaction  of 
mixed  saliva  is  usually  alkaline  but  may  on  fasting-,  also 
during  the  night  toward  morning,  and  2 — 3  hours  after 
meals,  or  after  much  talking,  become  acid.  It  also  becomes 
acid  on  standing  a  few  hours  (Repin).  It  is  more  or  less 
opalescent  and  viscid  and  foams  readily.  The  character  of 
the  saliva  will  vary  according  to  which  gland  furnishes  the 
most  of  the  secretion.  The  parotid  gland  yields  a  fluid 
secretion,  whereas  the  submaxillary  and  sub-lingual  glands 
yield  slimy  secretions.  In  febrile  diseases  the  secretion  of 
saliva  may  be  diminished  or  wholly  suppressed,  and  hence 
dryness  of  the  mouth  and  throat,  as  well  as  altered  taste. 
A  decrease  is  also  observed  in  diabetes,  in  severe  diarrhoeas, 
as  in  cholera.  The  administration  of  potassium  iodide  or 
of  mercury  produces  an  increased  flow,  or  salivation,  and 
the  composition  of  the  saliva  itself  becomes  altered.  Albu- 
min is  then  present  and  the  amount  of  salts  in  solution  is 
increased.  An  increased  flow  of  saliva  (ptyalism)  is  also 
brought  about  by  irritant  poisons  such  as  acids  and  alkalis; 
also  by  certain  foods,  lemon,  etc.,  and  occurs  also  in  some 
diseases,  especially  in  inflammatory  conditions  of  the  mouth, 
tonsils,  and  palate. 

In  icteric  conditions  the  saliva  does  not  contain  bile 
constituents.  In  diabetes  it  does  not  contain  sugar.  In 
the  latter  case,  however,  the  action  may  be  acid  because  of 
lactic  acid.  In  nephritis,  urea  maybe  present  in  the  saliva, 
and  uric  acid  has  been  found  in  uraemic  conditions.  Leucin 
has  been  found  in  the  saliva  of  a  hysteric  case. 


56  PHYSIOLOGICAL  CHEMISTRY. 

Salivary  calculi  which  are  occasionally  deposited  in 
the  salivary  ducts  consist  chiefly  of  calcium  carbonate  and 
phosphate,  cemented  with  organic  matter.  The  tartar  de- 
posited on  teeth  has  essentially  the  same  composition,  the 
phosphates  however  predominate.  These  calcium  salts  are 
held  in  solution  in  the  saliva  by  carbonic  acid.  On  expo- 
sure to  the  air  this  passes  off  and  the  salts  are  deposited. 

The  specific  gravity  of  the  mixed  saliva  varies  from  1.002 
to  1.008.  Such  saliva  contains  y2 — 1%  of  solids  which  consist 
of  albumin,  mucin,  ptyalin,  traces  of  urea  and  other  nitro- 
gen compounds  and  mineral  constituents.  The  amount  of 
saliva  secreted  in  the  course  of  24  hours  is  1400 — 1500  c.c. 
The  flow  is  increased  after  meals  and  by  pilocarpin.  Atro- 
pin  diminishes  salivary  secretion. 

The  chemical  examination  of  saliva  has  at  present  but 
little  clinical  significance.  Physiologically,  however,  the 
composition  and  action  of  saliva  is  of  the  greatest  import- 
ance. The  ferment  or  enzyme  present  in  the  saliva  is 
known  as  ptyalin  and  possesses  a  diastatic  or  amylolytic 
action.  That  is,  it  converts  starch  into  dextrin,  then  into 
iso-maltose  and  maltose.  Eventually  glucose  forms  prob- 
ably, however,  the  result  of  the  action  of  an  inverting  fer- 
ment. Ptyalin  is  not  present  in  the  saliva  of  all  animals. 
The  parotid  saliva  of  new-born  contains  ptyalin,  whereas 
the  submaxillary  saliva  does  not  contain  it  for  several 
months.  In  the  saliva  of  some  animals  as  the  horse  the 
ferment  is  not  present  in  the  free  state  but  as  a  zymogen 
from  which  it  readily  forms  in  mastication.  This,  as  well 
as  the  other  enzymes,  is  dragged  down  mechanically  by  a 
precipitate  of  calcium  phosphate  and  this  fact  is  utilized 
to  obtain  the  ferment  in  a  comparatively  pure  state. 

Although  ptyalin  resembles  in  its  action  the  diastase 
of  malt,  it  is  different.  This  is  seen  in  the  fact  that  the 
former  acts  best  at  40°,  the  latter  at  50 — 60°.  The  amount 
of  ptyalin  present  in  the  saliva  is  subject  to  variation. 
HC1  not  only  prevents  the  action  but  it  also  destroys  the 


SALIVA.  57 

ferment.     The   action   of   the   ptyalin   is   most   marked  in 
neutral  or  very  faintly  acid  saliva. 

A  microscopic  examination  of  the  saliva  will  always 
show  epithelial  cells  from  the  mouth  and  tongue,  also  sali- 
vary and  mucous  corpuscles.  Bacteria  are  always  numer- 
ous, and  certain  species  as  the  leptothrix,  spirillum,  and 
spirochsete  are  almost  invariably  present.  Among"  the 
pathogenic  forms  found  in  the  mouth  in  health  or  in  dis- 
ease may  be  mentioned  the  bacilli  of  diphtheria,  tubercu- 
losis and  tetanus,  Fraenkel's  diplococcus,  the  micrococcus 
tetragenus  and  the  pus-producing  staphylococci  and  strep- 
tococci, the  fungus  of  thrush  and  of  actinomycosis.  Blood 
or  pus  cells  may  be  present  in  the  saliva  in  inflammatory  or 
suppurative  conditions  of  the  mouth,  gums,  etc. 

Microscopic  Examination. — Rub  the  tongue  thoroughly 
over  the  inside  of  the  mouth,  teeth  and  gums,  collect  the 
saliva  and  examine  under  the  microscope  for  epithelial 
cells,  salivary  corpuscles,  etc. 

The  saliva  necessary  for  the  following  experiments  can 
be  readily  obtained  by  chewing  a  piece  of  pure  paraffin. 
Commercial  gum  must  not  be  used  inasmuch  as  it  con- 
tains sugar.     Collect  about  100  c.c.  of  saliva. 

1. — Test  the  reaction  of  the  mixed  saliva  with  litmus  paper. 
What  is  it? 

2.— Nearly  fill  a  50  c.c.  graduate  with  saliva.  If  there  is  any 
foam  on  the  surface  remove  it  with  a  piece  of  filter  paper.  Then 
immerse  an  urinometer  and  note  the  specific  gravity  of  mixed  saliva. 
What  is  the  reading"  if  immersed  in  pure  water? 

3. — To  about  5  c.c.  of  saliva  add  a  few  drops  of  acetic  acid  (1—10) 
and  gently  agitate.     A  flocculent  precipitate  of  mucin  forms. 

4. — To  some  saliva  apply  the  biuret  test  (Exp.  1,  p.  39).  The 
result  is  due  to  mucin. 

5. — To  some  saliva  add  a  drop  of  nitric  acid  and  boil.  Is  albumin 
present  in  saliva? 


58  PHYSIOLOGICAL  CHEMISTRY. 

6. — To  the  contents  of  the  tube  from  the  preceding"  experiment 
add  XH4OH.  An  orange  yellow  solution  forms— xanthoproteic  reac- 
tion. 

7. — To  some  saliva  add  a  few  drops  of  Millon's  reagent.  A  heavy- 
yellowish  precipitate  forms,  which  on  boiling  becomes  reddish.  This 
is  due  to  mucin. 

8. — To  some  of  the  saliva  add  a  drop  of  dilute  HC1,  then,  drop  by 
drop,  dilute  ferric  chloride  till  a  red  coloration  results.  This  is  due 
to  the  formation  of  ferric  sulphocyanate.  The  reaction  is  more  dis- 
tinct if  after  the  addition  of  HC1  the  liquid  is  filtered  and  the  ferric 
chloride  is  added  to  the  filtrate. 

9. — To  another  portion  of  saliva  add  a  little  iodic  acid  and  some 
starch  solution.  Iodine  is  liberated  and  colors  the  starch  blue.  This 
is  due  to  a  sulphocyanate.     Explain  the  reaction. 

10. — To  some  saliva  add  a  few  drops  of  dilute  H2S04,  mix;  then 
add  a  few  drops  of  a  colorless  solution  of  potassium  iodide  and  finally 
a  few  drops  of  starch  solution.  Iodine  is  liberated  and  colors  the 
starch  blue.     This  is  due  to  nitrous  acid.     Explain. 

11. — To  some  saliva  add  a  drop  or  two  of  dilute  HC1,  then  2 — 3 
drops  of  a  saturated  sulphanilic  acid  solution  and  mix.  Now  add  a 
few  drops  of  naphthylamine  hydrochloride.  A  pink  or  red  solution 
indicates  the  presence  of  nitrous  acid.  This  test  is  employed  in  test- 
ing for  nitrites  in  water  analysis. 

12. — Take  a  small  dose  of  potassium  iodide,  rinse  out  the  mouth 
thoroughly  with  water,  and  test  some  of  the  saliva  at  once  for  KI. 
This  is  done  by  adding  to  some  of  the  saliva  a  little  chlorine  water, 
and  then  shaking  with  carbon  bi-sulphide.  A  pink  coloration  of  the 
latter  indicates  iodine.  Iodine  should  be  absent  from  the  saliva  after 
rinsing.  After  that  collect  a  little  of  the  saliva  every  15  minutes, 
and  test  for  iodine  as  above.  How  soon  does  KI  appear  in  the  saliva 
after  being  taken  into  the  stomach? 

13. — Separation  of  Mucin. — Pour  10  c.c.  of  the  saliva  slowly  and 
with  constant  stirring,  into  50  c.c.  of  absolute  alcohol.  A  fibrinous 
light  precipitate  forms.  Allow  to  settle  over  night  in  a  covered 
beaker.  Then  filter,  wash  the  precipitate  on  the  filter  twice  with 
alcohol,  then  with  ether.  Spread  out  the  filter  to  dry  and  finally  with 
a  spatula  remove  the  white  chalky  powder  of  mucin. 

(a).  To  a  little  of  the  powdered  mucin  in  a  tube  add  some  water. 
It  swells  up,  but  does  not  pass  into  solution.     Then  add  a  drop  or  two 


SALIVA.  59 

of  KOH  when  it  dissolves,  forming  a  milky  solution.     To  this  solution 
now  apply  the  biuret  test.     What  is  the  result? 

(b).  Place  the  remainder  of  the  powder  in  a  tube  and  add  dilute 
HC1  (1 — 3)  and  boil  for  some  minutes.  Transfer  a  portion  to  another 
tube,  cool,  render  alkaline  with  KOH  and  boil  with  Fehling's  solution. 
The  formation  of  red  cuprous  oxide  indicates  the  presence  of  a 
reducing-  substance.  If  this  test  is  not  given,  boil  again  the  remain- 
ing original  liquid,  and  again  test  a  portion  as  above. 

Mucin  is  a  complex  proteid  substance  and  on  decompo- 
sition, as  above,  it  yields  a  reducing-  compound  which,  how- 
ever, is  not  sugar.  What  other  substances  on  heating  with 
an  acid  yield  reducing  substances? 

14. — Action  of  Ptyalin. — Prepare  a  starch  solution  according  to 
the  directions  given  under  starch  (p.  28,  Exp.  3).  Into  each  of  eight 
tubes  place  about  3  c.c.  of  Fehling's  solution.  Into  each  of  another 
set  of  eight  tubes  place  1 — 2  drops  of  dilute  iodine  solution. 

(a).  To  30  c.c.  of  the  starch  solution  in  a  graduate  add  six  drops 
of  saliva,  and  at  once  mix  thoroughly.  Immediately  after  mixing 
pour  2 — 3  c.c.  of  the  mixture  into  one  of  the  tubes  containing  Fehl- 
ing's solution,  and  also  a  portion  into  a  tube  containing  the  iodine. 
The  latter  colors  deep  blue — due  to  starch.  Boil  the  tube  with  Fehl- 
ing's solution.     No  reaction  should  take  place — absence  of  sugar. 

At  intervals  of  two  minutes  apply  the  test  with  Fehling's  solu- 
tion and  with  iodine  to  the  mixture  in  the  manner  just  given.  Tabu- 
late your  results,  noting  the  time  when  sugar  appears  in  the  mixture; 
when  erythro-dextrin  and  achroo-dextrin  appear.  The  time  of  ap- 
pearance of  the  latter  is  spoken  of  as  the  achromic  point.  When 
this  is  reached  boil  some  of  the  starch  mixture  with  Barfoed's  reagent. 
What  is  the  result?    What  does  this  indicate? 

At  the  conclusion  of  this  experiment  add  to  each  of  the  iodine 
tubes  5  c.c.  of  water.  The  characteristic  color  of  the  starch  and  of  the 
several  dextrins  will  be  more  apparent.  Complete  conversion  should 
take  place  in  about  15  minutes.  If  it  does  not,  repeat  the  experiment 
using  a  larger  amount  of  saliva. 

b  .  To  10  c.c.  of  starch  solution  add  5  c.c.  of  saliva,  mix  and  make 
tests  as  rapidly  as  possible  with  iodine  and  with  Fehling's  solution. 
What  is  the  result? 

(c).  Boil  5  c.c.  of  saliva  in  a  tube  for  1 — 2  minutes,  then  add  10 
c.c.  of  the  starch  solution  and  mix.     Immediately  test  a  portion  as 


60  PHYSIOLOGICAL   CHEMISTRY. 

above  and  also  at  the  end  of  15  minntes.     What  is  the  result?    What 
is  the  action  of  heat  on  ptyalin? 

{d).  To  10  c.c.  of  starch  solution  add  0.2  c.c.  of  a  1%  acetic  acid 
solution,  mix  and  then  add  two  drops  of  saliva.  Test  immediately 
with  iodine  and  with  Fehling's  solution,  and  also  at  the  end  of  5,  10, 
and  15  minutes.  The  mixture  contains  about  0.02%  acetic  acid. 
What  is  the  effect  of  this  amount  of  acetic  acid  on  the  rate  of 
inversion? 

(e).  To  10  c.c.  of  the  starch  solution  add  0.6  c.c.  of  a  dilute  HC1 
(0.3%).  The  latter  is  prepared  by  adding  8  c.c.  of  the  concentrated 
acid  to  one  litre  of  water.  Mix,  then  add  two  drops  of  saliva 
and  again  mix  thoroughly.  This  mixture  now  contains  about  0.02% 
HC1,  about  the  same  degree  of  acidity  as  in  the  preceding  experi- 
ment, and  about  one-tenth  of  that  of  the  gastric  juice.  Test  the 
mixture  at  once  with  iodine  and  with  Fehling's  solution,  and  also  at 
the  end  of  5,  10,  and  15  minutes.  Note  carefully  the  result.  How  does 
the  action  of  HC1  compare  with  that  of  acetic  acid. 


.    CHAPTER    V. 
GASTRIC  JUICE. 

The  gastric  juice  is  the  combined  product  of  several 
glands,  cardiac  and  pyloric,  and  normally  possesses  an 
intense  acid  reaction  due  to  the  presence  of  free  hydro- 
chloric acid.  In  certain  diseases  as  a  result  of  fermenta- 
tive changes  the  acidity  may  be  in  part,  or  wholly,  due  to 
organic  acids-such  as  lactic,  acetic,  butyric,  etc.  The 
material  which  is  obtained  from  the  stomach  after  a  test 
breakfast,  by  means  of  lavage,  usually  contains  besides 
remnants  of  food,  more  or  less  mucus.  Before  it  is  em- 
ployed for  tests  it  should  therefore  be  filtered.  The  gas- 
tric juice  proper  is  a  watery  fluid  which  filters  easily  and 
is  not  slimy.     Its  specific  gravity  varies  from  1,002  to  1,010. 

The  contents  of  the  stomach  ,may  contain:  (1)  Micro- 
scopical constituents,  such  as  remains  of  food;  squamous 
epithelial  cells,  rarely  columnar;  blood  cells;  various  micro- 
organisms, such  as  bacilli,  micrococci,  rarely  spirals;  sar- 
cines,  yeasts  and  leptothrix  threads  are  common.  (2)  Sol- 
uble chemical  constituents. 

The  latter  are  of  first  importance  physiologically. 
They  include  the  proteolytic  enzyme,  pepsin;  the  milk- 
curdling  ferment,  rennin  or  chymosin;  hydrochloric  acid 
present  either  in  free  state,  or  loosely  combined,  or  as 
ordinary  chloride;  organic  acids,  such  as  lactic  acid,  etc., 
resulting  from  bacterial  fermentation;  lastly  acid  phos- 
phates, peptons,  etc. 

The  secretion  from  the  pyloric  end  of  the  stomach  is 
usually  said  to  be  alkaline  and  to  contain  only  pepsin.  On 
the  other  hand,  the  secretion  from  the  cardiac  end  is  in- 
tensely acid  and  contains  pepsin. 


62  PHYSIOLOGICAL  CHEMISTRY. 

The  hydrochloric  acid  is  unquestionably  derived  from 
the  sodium  chloride  of  the  blood.  As  a  result  of  mass 
action  the  large  amount  of  carbon  dioxide  present  in  the 
blood  reacts  upon  the  sodium  chloride.  Dissociation  re- 
sults and  a  minute  amount  of  free  hydrochloric  acid  is 
present  in  the  blood.  Certain  cells  of  the  stomach  by  vir- 
tue of  their  selective  power  transmit  this  free  acid  to  the 
gastric  juice. 

The  amount  of  free  hydrochloric  acid  present  in  the 
gastric  secretion  varies  considerably,  but  in  general  is  said 
to  average  about  0.2  per  cent.  On  contact  with  albuminous 
substances  the  acid  unites  with  these  and  gives  rise  to 
what  is  known  as  "loosely-combined  hydrochloric  acid." 
When  in  this  combination  the  protein  molecule  is  prepared 
for  the  action  of  the  ferment  pepsin.  Consequently  the 
loosely  combined  hydrochloric  acid  may  be  considered  as 
the  physiologically  active  acid.  Furthermore,  the  .hydro- 
chloric acid  probably  unites  with  pepsin  to  form  the  so- 
called  "pepsin-hydrochloric  acid." 

Hydrochloric  acid  is  an  effective  germicide  and,  more- 
over, stops  the  diastatic  action  of  ptyalin  on  starch.  It 
does  not  follow,  however,  that  the  salivary  digestion  of 
starch  ceases  the  moment  the  food  reaches  the  stomach,  or 
that  all  bacteria  are  destroyed  in  the  stomach.  With  refer- 
ence to  starch  conversion  it  may  be  said  that  this  will  con- 
tinue until  free  hydrochloric  acid  has  permeated  the  entire 
mass  of  food  contained  in  the  stomach.  If  the  mass  of  food 
is  large,  or  if  the  amount  of  hydrochloric  acid  secreted  is 
small,  this  diastatic  action  may  continue  for  a  considerable 
time  after  the  food  has  been  taken  into  the  stomach. 

As  to  the  germicidal  action  of  the  hydrochloric  acid 
contained  in  the  gastric  juice  it  should  be  remembered  that 
it  is  exerted  against  the  vegetative  form  of  bacteria  and 
not  against  spores.  Moreover,  since  ptyalin  may  continue 
to  act  on  starch  in  the  stomach  for  some  time  owing  to  the 
fact  that  the  acid  secreted  must  first  neutralize  basic  con- 


GASTRIC  JUICE.  .  63 

stituents,  unite  with  proteins,  and  finally  permeate  the 
entire  mass  as  free  acid,  it  follows  that  many  bacteria 
introduced  with  the  food  may  not  be  exposed  to  the  free 
acid  for  a  considerable  period  of  time.  Portions  of  the 
stomach  contents  are  passed  into  the  intestines  at  frequent 
intervals,  and  hence  even  weak,  vegetative  forms,  such  as 
the  cholera  vibrio  may  pass  the  stomach  uninjured.  It  is  not 
necessary  to  neutralize  the  stomach  contents  in  man  and  in 
animals  in  order  to  produce  experimental  cholera  infection. 

An  increased  secretion  of  hydrochloric  acid  (hyper- 
acidity) results  in  more  rapid  solution  of  proteins,  and  in 
early  inhibition  of  the  diastatic  action  of  saliva  on  starch. 
Such  a  stomach  fluid  though  rich  in  free  acid,  if  it  remains 
in  the  stomach  for  some  time,  undergoes  bacterial  decom- 
position. Organic  acids,  gases,  as  marsh-gas,  hydrogen, 
carbon  dioxide,  etc.,  form,  but  putrefaction  of  the  albu- 
minous substances  does  not  take  place. 

Hydrochloric  acid  is  frequently  diminished  in  amount 
(hypo-  or  sub-acidity).  This  decrease  may  be  considera- 
ble, but  the  total  absence  of  free  hydrochloric  acid  is  rare. 
Such  a  decrease  is  met  with  commonly  in  dyspepsia,  in 
cancer  of  the  stomach,  in  febrile  diseases,  in  cirrhosis  of 
the  liver  and  frequently  in  nephritis. 

With  the  decrease  in  the  amount  of  free  acid  present, 
naturally  its  inhibiting  influence  on  bacteria  is  diminished, 
consequently  bacterial  growth  in  the  stomach  may  become 
enormously  developed.  In  such  instances  even  the  charac- 
teristic mouth  bacteria  as  leptothrix,  spirochetes,  comma 
bacilli,  etc.,  may  develop  in  the  stomach  contents.  As  a 
result  then,  of  a  decrease  in  free  hydrochloric  acid,  various 
fermentations  may  arise.  At  one  time  it  may  be  lactic 
acid,  at  another  time  acetic  or  butyric  acid,  etc.,  that  is 
especially  being  elaborated.  The  longer  the  food  remains 
in  the  stomach,  because  of  motor  insufficiency,  the  more 
marked  will  such  decomposition  be. 

A  stomach  will  undoubtedly  disinfect  itself  if  a  suffi- 


'64  .  PHYSIOLOGICAL  CHEMISTRY. 

cient  interval  is  allowed  between  meals.     These  intervals 
should  be  not  less  than  10  or  12  hours. 

The  enzymes,  pepsin  and  rennin,  are  secreted  by  the 
stomach  even  in  severe  diseases,  and  it  is  only  in  general 
atrophy  of  the  mucous  membrane  of  the  stomach  that  they 
are  said  to  be  absent.  It  is  doubtful,  however,  if  these 
enzymes  are  ever  wholly  absent  from  the  gastric  secretion. 
Pepsin,  as  well  as  a  diastatic  ferment,  appear  in  the  urine 
in  starvation.  These  ferments  are  frequently  increased  in 
the  urine  in  fevers,  as  typhoid  fever,  tuberculosis,  pneu- 
monia, etc.,  and  are  markedly  increased  in  diabetes.  In 
testing-  urine  for  enzymes  the  utmost  care  must  be  taken  to 
eliminate  the  action  of  bacteria. 

Pepsin  is  present  in  the  gastric  secretion  of  all  verte- 
brates. It  acts  on  proteins  in  acid,  but  not  in  neutral  or 
alkaline  solution.  It  has  no  action  on  fats,  or  on  carbo- 
hydrates. There  is  reason  to  believe  that  the  pepsin  of 
one  animal  is  different  from  that  of  another.  In  other 
words,  that  there  is  a  group  of  pepsins,  or  proteolytic  fer- 
ments just  as  there  is  a  group  of  nucleins,  of  haemoglobins, 
etc.  Similar  proteolytic  ferments  are  met  with  in  plants 
(papayotin,  etc).  Pepsin,  when  isolated  in  the  purest  con- 
dition possible,  does  not  give  most  of  the  protein  reactions. 
When  impure  it  is  soluble  in  water  and  in  glycerin.  It  is 
precipitated  by  alcohol,  though  this  precipitation  is  to  be 
considered  largely  as  a  mechanical  dragging-  down  of  the 
enzymes,  as  in  similar  precipitation  of  bacterial  toxins. 
Pepsin,  when  in  solution,  is  readily  destroyed  by  heat,  even 
at  55 — 60°;  whereas  when  dry  it  can  be  heated  to  above 
100°  without  change. 

The  characteristic  action  of  pepsin  is  its  digestion  of 
proteins  in  acid  solutions.  Nucleoproteids  on  digestion 
with  pepsin  leave  a  residue  of  nuclein  (or  pseudo-nuclein  in 
the  case  of  certain  nucleo-albumins  such  as  casein).  Even 
fibrin  leaves  a  residue  of  nuclein  on  digestion  which  has 
been  called  dyspepton. 


GASTRIC  JUICE.  65 

The  albuminous  substances  are  first  brought  into  solu- 
tion by  the  free  hydrochloric  acid.  If  this  solution  is  neu- 
tralized a  precipitate  of  syntonin  or  acid  albumin  forms. 
As  a  result  of  the  action  of  the  ferment  the  protein  is  con- 
verted into  albumose.  It  is  only  when  peptic  digestion  is 
protracted,  as  for  some  days,  that  the  albumoses  in  turn 
are  changed  into  peptons.  In  peptic  digestion  the  cleavage 
does  not  go  below  the  pepton  stage,  whereas  in  pancreatic 
digestion  a  portion  of  the  pepton  formed  may  be  split  up 
into  leucin,  tyrosin,  etc. 

The  albumoses  and  peptons  are  to  be  considered  as 
hydration  products  of  proteins,  corresponding  to  the  dex- 
trins  and  to  glucose  in  the  hydration  of  starches.  Just  as 
the  hydration  of  starches,  fats,  etc.,  may  result  from  the 
action  of  enzymes,  or  on  heating  with  acids  or  alkalis,  or 
by  bacterial  ferments,  so  the  hydration  of  proteins  may  be 
accomplished  by  these  same  agencies.  As  a  result  of  the 
action  of  bacteria,  especially  of  the  liquefying  type,  albu- 
min or  fibrin  may  be  changed  to  albumose,  or  even  to  pep- 
ton. The  bacterial  proteids  probably  owe  their  poisonous 
properties  to  a  mere  admixture  of  the  real  toxin.  Poison- 
ous proteids  (abrin,  ricin)  have  been  isolated  from  plants, 
and  also  from  the  venoms  of  serpents. 

The  first  product  of  the  hydration  of  proteins  is  an 
albumose,  the  final  product  is  pepton.  Strictly  speaking 
there  is  not  merely  one  albumose,  but  a  group  of  albu- 
moses. The  following  four  types  of  albumose  are  dis- 
tinguished: 

Hetero- albumose  is  insoluble  in  water,  but  soluble  in 
dilute  sodium  chloride.  Proto-albumose  is  soluble  in  water 
and  in  sodium  chloride.  Both  of  these  are  precipitated  by 
sodium  chloride  in  neutral  solution,  though  incompletely. 
They  are  sometimes  designated  as  primary  albumoses.  As 
a  result  of  the  action  of  salts,  etc. ,  hetero-albumose  may 
become  insoluble  and  is  then  known  as  dys- albumose. 


t)6  PHYSIOLOGICAL   CHEMISTRY. 

Deutero-albumose  is  soluble  in  water  and  in  dilute  sodium 
chloride,  but  is  not  precipitated  from  neutral  solution  on 
saturation  with  sodium  chloride.  It  is,  however,  precipi- 
tated incompletely  out  of  acid  solution.  This  albumose  is 
closely  related  to  pepton  and  is  sometimes  spoken  of  as 
secondary  albumose. 

Not  only  are  there  several  albumoses  resulting  from 
the  digestion  of  a  given  protein,  but  when  these  are  com- 
pared with  similar  products  formed  from  the  digestion  of 
other  proteins,  certain  differences  are  manifest.  It  is  cus- 
tomary to  designate  the  albumoses  formed  from  globulin, 
vitellin,  casein,  myosin,  as  globulinose,  vitellose,  caseose, 
myosinose,  respectively.  The  word  proteose  is  used  as  a 
general  term,  covering  all  albumoses  whether  derived  from 
animal  or  vegetable  protein  matter. 

Pepton  is  very  hygroscopic  and  very  soluble  in  water. 
It  diffuses  more  readily  than  does  albumose.  It  is  not  pre- 
cipitated by  picric  acid  or  by  potassium  mercuric  iodide; 
and  is  incompletely  precipitated  by  phosphotungstic  and 
by  phosphomolybdic  acids.  It  is  precipitated  by  tannic 
acid,  but  the  precipitate  is  soluble  in  excess. 

The  protein  molecule  on  hydration  yields  two  groups  of 
compounds,  one  of  which  is  more  resistant  than  the  other. 
The  more  resistant  product  is  designated  as  anti-albumose, 
anti- pepton,  whereas  the  less  resistant  product  is  termed 
hemi-albumose,  hemi-pepton.  The  gastric  juice  when 
allowed  to  act  for  some  days  yields  a  mixture  of  anti-  and 
hemi-pepton.  This  mixture  is  known  as  ampho-pepton 
If  this  is  subjected  to  a  more  energetic  ferment  than  pep- 
sin, namely  trypsin,  the  hemi-pepton  is  broken  down  into 
leucin  and  tyrosin,  whereas  anti-pepton  remains  consti- 
tuting one-half  of  the  original  mixed  peptons. 

It  should  not  be  inferred  from  the  preceding  state- 
ment that  all  the  hemi-pepton,  under  the  influence  of  trypsin 
in  ihe  intestine,  is  broken  down  into  leucin  and  tyrosin.     It 


GASTRIC  JUICE.  67 

does  undergo  this  decomposition  in  vitro,  but  it  does  not 
follow  from  this  that  the  same  complete  cleavage  results 
in  the  intestines.  The  fact  that  leucin  and  tyrosin  are 
present  in  the  intestinal  contents  in  minimal  amounts,  and 
the  further  important  fact  that  anti-pepton  alone  will  not 
support  life,  indicate  that  hemi-pepton  is  absorbed  and 
utilized.  The  hemi-  and  anti-peptons  are  regenerated  to 
albumin,  etc. 

(  Anti-albumose —  Anti-pepton      ) 
Proteins  -  [■  Ampho-pepton. 

f  Hemi-albumose — Hemi-pepton   )  (leucin,  tyrosin). 

Antipepton  possesses  the  formula  C10H15N3O5,  and  is 
identified  with  sarkinic  acid,  derived  from  phosphosarkinic 
acid  of  the  muscle.  It  gives  a  strong  biuret  reaction  in  the 
cold,  but  does  not  give  Millon's  test. 

When  a  haemorrhage  into  the  stomach  takes  place  the 
haemoglobin  is  acted  upon  by  the  gastric  juice  and  is  split 
into  haematin  and  globulin.  The  former  is  the  cause  of  the 
brown,  or  coffee  color,  of  the  stomach  contents. 

The  milk  curdling  ferment,  known  as  rennin  or  chymo- 
sin,  is  apparently  a  constant  constituent  of  the  gastric 
juice  of  vertebrates.  It  is  especially  abundant  in  the 
mucous  membrane  of  the  stomach  of  the  calf  (rennet),  and 
this  is  used  to  curdle  milk  in  the  manufacture  of  cheese. 
It  may  be  absent  in  cancer  and  in  chronic  catarrh  of  the 
stomach,  and  in  atrophy  of  the  mucous  membrane  of  the 
stomach.  When  obtained  in  the  purest  condition  possible 
it  does  not  give  the  ordinary  protein  reactions.  It  is  de- 
stroyed by  0.3  per  cent.  HC1  in  the  presence  of  pepsin.  It 
coagulates  milk  or  solutions  of  casein  containing  lime  salts. 
Calcium  is  necessary  for  the  curdling  of  milk  and  for  the 
coagulation  of  blood.  In  the  latter  instance  the  calcium  is 
necessary  in  order  to  make  fibrin  ferment;  in  the  former 
case  it  unites  with  the  altered  casein  to  make  para-casein. 


68  PHYSIOLOGICAL  CHEMISTRY. 

I.     Recognition  of  Free  Hydrochloric  Acid. 

Three  solutions  of  dilute  HC1  labelled  1,  2,  and  3,  will  be  found  on 
the  side  table.  Solution  1  approximates  in  strength  that  found  in 
the  gastric  juice.  It  is  prepared  by  diluting  6  c.c.  of  HC1  (1.19  specific 
gravity)  to  one  liter  (=  0.25% ).  Solution  2  is  prepared  by  diluting  200 
c.c.  of  solution  1  to  one  liter  (—  0.05%).  Solution  3  is  prepared  by 
diluting  200  c.c.  of  solution  2  to  one  liter  (=  0.01%). 

A  2%  pepton  and  a  1%  lactic  acid  solution  will  also  be  found  on 
the  side  table.  If  the  pepton  solution  is  slightly  alkaline  it  should  be 
faintly  acidified  with  acetic  or  lactic  acid. 

The  following-  tests  are  given  in  the  order  of  their 
delicacy: 

1. — Bi-methyl-amido-azobenzol. — This  reagent  is  used  in  a  0.5%  alco- 
holic solution.  Add  3 — 4  drops  of  the  reagent  to  some  of  the  solution 
to  be  examined.  If  a  pink  red  color  forms  a  free  mineral  acid  is 
present.  In  the  case  of  gastric  juice  it  is  HC1.  A  yellow  color  indi- 
cates an  absence  of  HC1.  Certain  substances,  such  as  pepton  and 
organic  acids,  tend  to  interfere  in  this  as  well  as  in  the  subsequent 
tests.  Organic  acids,  if  concentrated,  may  give  a  somewhat  similar 
reaction. 

Note  the  results  obtained  with  solutions  1,  2,  and  3,  in  the  first 
column.  Then  mix  the  same  amount  of  these  solutions  with  an  equal 
volume  of  2%  pepton,  and  to  this  mixture  apply  the  test  and  note  the 
results  in  the  second  column.  In  the  same  way  make  a  mixture  of 
the  three  solutions  with  an  equal  volume  of  a  1%  solution  of  lactic 
acid,  test  and  note  the  results. 


1  c.c.  of  solution  1 
lc.c.  "  "  2 
lc.c.  "  "  3 
Limit  of  delicacy. 


Pure  HC1. 


HC1  and 
2%  Pepton  aa, 


HC1  and 
Lactic  Acid  aa. 


Apply  the  test  to  the  solution  of  pepton,  also  to  the  solution  of 
lactic  acid.     Report  the  results. 

2. — Gunzburg's  reaction,  or  the  phloi-oglucin  vanillin  test. — The  rea- 
gent is  prepared  by  dissolving  1  g.  of  vanillin  and  2  g.  of  phloroglucin 
in  100  c.c.  of  alcohol. 


GASTRIC  JUICE.  69 

Place  the  solution  to  be  tested  in  an  evaporating-  dish,  add  2 — 3 
drops  of  the  reagent  and  carefully  evaporate  over  a  small  flame  to 
dryness.  A  purple  or  pinkish-red  color  indicates  free  HC1.  This  has 
long  been  considered  the  most  delicate  test  for  free  HC1. 

Apply  this  test  to  solutions  1,  2  and  3,  and  to  mixtures  as  indi- 
cated in  the  table  given  under  experiment  1.  Carefully  note  the 
limit  of  delicacy  of  the  reaction  under  the  several  conditions.  Tabu- 
late the  results  as  above. 

3. — Boas'  reagent. — This  is  prepared  by  dissolving  10  g.  of  resorcin, 
3  g.  of  cane  sugar  and  3  c.c.  of  alcohol  in  100  c.c.  of  water.  Place  in 
an  evaporating  dish  the  solution  to  be  tested,  add  2 — 3  drops  of  the 
reagent,  and  evaporate  over  a  small  flame  to  dryness.  If  a  free 
mineral  acid  is  present  a  rose  or  pink-red  color  develops  and  gradually 
fades  on  cooling. 

Apply  this  test  to  solutions  1,  2  and  3,  and  to  mixtures  as  indi- 
cated in  the  table  given  in  experiment  1.  Note  the  limit  of  delicacy 
and  tabulate  the  results. 

4. — Tropceolin  00. — A  solution  of  this  reagent  is  prepared  by  dis- 
solving 0.25  g.  of  the  reagent  in  1,000  c.c.  of  water.  Instead  of  the 
solution  tropaeolin  papers  may  be  employed.  They  are,  however,  not 
so  reliable  as  the  solution,  since  with  distilled  water  they  sometimes 
give  a  pink  color.  To  some  of  the  acid  solution  add  a  drop  of  the 
reagent  (or  immerse  a  strip  of  the  tropaeolin  paper).  A  pink  color  is 
due  to  free  mineral  acid.  If  the  solution  is  evaporated  carefully  to 
dryness  a  bluish  residue  remains. 

Apply  this  test  to  the  solutions  as  given  in  experiment  1,  and 
tabulate  the  results. 

5. — Congo-red  papers. — Tne  color  of  these  papers  is  changed  on 
contact  with  mineral  acids  to  a  deep  blue,  whereas  organic  acids 
yield  a  violet.  Immerse  a  strip  of  the  paper  in  1  c.c.  of  the  solutions 
1,  2  and  3,  also  lactic  acid  and  distilled  water,  and  report  the  results 
and  the  delicacy. 

6. — Benzopv/rpwrin  6  11  i><<\><  /■*.— These  papers  are  turned  to  an 
intense  dark  brown  color  by  mineral  acids.  With  strips  of  this  paper 
make  similar  tests  as  those  given  in  experiment  5,  and  report  the 
results. 

7. — Methyl  violet. — A  solution  of  this  reagent  is  prepared  by  dis- 
solving 0.5  g.  in  1,000  c.c.  of  water.  To  the  solution  to  be  tested  add 
1—2  drops  of  the   reagent.      Free  HC1  gives  a  copper-blue    color. 


70  PHYSIOLOGICAL  CHEMISTRY. 

Organic  acids  yield  a  violet  blue.  Apply  this  test,  first  to  some  dis- 
tilled water,  and  note  the  color.  Finally  apply  the  test  to  the  solu- 
tions 1,  2  and  3,  and  compare  the  results.  Also  test  pepton  and  lactic 
acid  mixtures  and  tabulate  the  results  as  under  experiment  1. 

II.     Detection  of  Lactic  Acid. 

Uffelmann's  test. — The  reagent  is  prepared  by  adding  a  drop  of 
dilute  ferric  chloride  to  10  c.c.  of  a  2.5%  carbolic  acid  solution.  The 
liquid  is  colored  blue.  This  color  is  completely  discharged  by  mineral 
acids,  leaving  a  colorless  solution,  whereas  organic  acids  discharge 
the  color  and  leave  a  straw  yellow  solution.  A  1%  lactic  acid  solu- 
tion is  used. 

1. — In  each  of  three  test-tubes  place  5  c.c.  of  the  reagent,  then 
add  to  each  about  Yz  c.c.  of  the  lactic  acid  solution.  The  blue  is 
replaced  by  a  straw  yellow  color. 

To  each  of  these  tubes  now  add  respectively  an  equal  volume  of 
the  HC1  solutions  1,  2  and  3.  Note  the  interference,  if  any,  in  the 
lactic  acid  reaction  by  the  presence  of  variable  amounts  of  free  HC1. 

2. — -In  each  of  three  tubes  place  5  c.c.  of  the  reagent,  then 
add  respectively  an  equal  volume  of  the  HC1  solutions  1,  2  and  3. 
Compare  the  results  with  those  obtained  above  when  lactic  acid  is 
present. 

3. — In  each  of  six  tubes  place  5  c.c.  of  an  almost  colorless  solu- 
tion of  FeCl3.  To  tube  1  add  1  c.c.  of  the  HC1  solution  1.  To  tube  2 
add  1  c.c.  of  the  lactic  acid  solution.  To  tube  3  add  1  c.c.  of  the  2% 
pepton  solution.  To  tube  4  add  1  c.c.  of  alcohol.  To  tube  5  add  1  c.c. 
of  a  4%  solution  of  cane  sugar.  Tube  6  remains  blank  and  serves  for 
a  control.     Carefully  note  the  results. 

It  is  evident  from  the  above  experiments  that  this  test 
for  lactic  acid  is  not  characteristic.  In  the  first  place  free 
HC1  if  present  in  sufficient  amount  may  interfere;  and  sec- 
ondly, a  similar  test  is  given  by  a  number  of  substances 
which  may  at  times  be  present  in  the  stomach  contents. 
In  order  to  obtain  a  positive  test  for  lactic  acid  it  is  neces- 
sary to  isolate  the  lactic  acid  from  the  liquid  by  extraction 
with  ether.     The  liquid   must  be  extracted  several  times 


GASTRIC  JUICE. 


11 


with  ether.     This  is  then  distilled  off,  the  residue  dissolved 
in  water  and  tested  as  above. 


III.     Peptic  Digestion. 

The  following- solutions  will  be  found  on  the  side  table: 

l.—A  0.25%  solution  ofHCl.—This  solution  is  the  same  as  solution 
1,  used  in  connection  with  the  tests  for  free  HC1.  It  corresponds  to 
the  normal  acidity  of  the  gastric  juice. 

2— A  solution  of  pepsin  in  water.— This  is  prepared  by  dissolving 
I  1  g.  of  pepsin  in  1,000  c.c.  of  water. 

3.-^4  pepsin-hydrochloric  acid  solur 
Hon.—  This  is  prepared  by  dissolving- 
1  g.  of  pepsin  in  1  liter  of  solution  1. 

Label  six  tubes  and  equip  as 
follows: 

1.— In  tubes  1  and  2  place  20  c.c. 
of  solution  3  and  about  2  g.  of  fresh 
washed  fibrin.  Too  much  fibrin  should 
be  avoided. 

Place  in  tube  3,  10  c.c.  of  solu- 
tion 3.  Immerse  in 
boiling  water  for 
about  2—3  minutes; 
then  cool  to  40°,  and 
add  2  g.  of  fibrin. 

Place  in  tube  4, 
10  c.c.  of  solution  1 
and  about  2  g.  of 
fibrin. 

Place  in  tube  5, 
10  c.c.  of  solution  2 
and    about    2    g.    of 
fibrin. 
Place  in  tube  6,  2.5  c.c.  of  solution  3  and  7.5  c.c.  of  solution  1 
Then  add  about  2  g.  of  fibrin. 

The  tubes  thus  prepared  are  placed  in  an  incubator  at  40°  or 
immersed  in  a  water-bath  having  that  temperature  (Fig.  2).     At  the 


72  PHYSIOLOGICAL,  CHEMISTRY. 

end  of  15  minutes  the  tubes  are  taken  out  and  examined.  Observe 
that  in  all  the  tubes,  except  tube  5,  the  fibrin  has  swelled  up  so  that 
the  contents  of  the  tube  are  solid.  Return  the  tubes  to  the  incubator 
or  water-bath  and  examine  at  the  end  of  every  hour  for  the  next 
three  hours.  Observe  the  change  that  takes  place  in  tubes  1  and  2, 
and  compare  carefully  with  tubes  3,  4  and  5,  and  with  tube  6. 

The  tubes  remain  in  the  incubator  till  next  day.  If,  however, 
tube  1  is  completely  digested  in  2 — 3  hours,  it  should  be  treated  at 
once  according  to  experiment  2.  Next  day  carefully  examine  and 
note  the  condition  of  each  tube.  In  tubes  1  and  2,  and  possibly  in 
tube  6,  the  fibrin  has  disappeared.  A  finely  granular,  whitish  or 
brownish  sediment  is  left.  What  is  it?  Tubes  3  and  4  are  about 
alike.  The  fibrin  is  gradually  being  dissolved  by  the  dilute  acid.  The 
pepsin  added  to  tube  3  evidently  has  been  destroyed  by  boiling.  No 
change  in  tube  5. 

Return  tubes  2,  3  and  4,  to  the  incubator,  and  keep  there  for  3 — 4 
days  longer. 

2. — Filter  the  contents  of  tube  1.  Save  a  portion  of  the  filtrate 
for  Exp.  B. 

(A).  To  10  c.c.  of  the  filtrate,  in  a  small  beaker,  or  in  a  wide  test- 
tube  on  foot,  add  8  g.  of  powdered  (NH4)2S04.  Immerse  for  %—l  hour 
in  a  water-bath  at  a  temperature  of  30 — 35°.  Stir  frequently  with 
a  rod  to  bring  the  salt  into  solution.  When  the  salt  ceases  to  dis- 
solve, i.  e.,  when  the  liquid  is  saturated,  the  albumose  present  will  be 
thrown  out  of  solution  as  coarse  floccules  which  rise  to  the  surface 
forming  a  sticky  or  slimy  layer.  Transfer  the  liquid  to  a  filter  pre- 
viously moistened  with  a  little  saturated  (NR~4)2S04  solution.  Wash 
the  residue  with  10  c.c.  of  saturated  (NH4)2S04  solution. 

(a).  The  clear  (NH4)2S04  filtrate  contains  pepton.  Test  this 
solution  as  follows: 

1. — To  a  little  of  the  liquid  add  an  equal  volume  of  strong  NaOH, 
then  1 — 2  drops  of  very  dilute  CuS04  solution.  A  pink  color  results. 
The  biuret  test  is  given  in  the  cold  by  the  hydrated  proteids. 

2. — To  another  portion  of  the  filtrate  add  1 — 2  drops  of  a  fresh 
tannic  acid  solution.  Avoid  an  excess  of  reagent.  A  heavy  white 
precipitate  forms. 

3. — To  a  portion  add  one  to  two  drops  of  dilute  acetic  acid,  then 
a  drop  or  two  of  potassium  f  errocyanide.  What  does  the  absence  of 
a  precipitate  mean? 


GASTRIC  JUICE.  73 

{b).  The  (NH4)2S04  precipitate  left  on  the  filter  is  albumose. 
Transfer  to  a  tube,  add  distilled  water,  warm  gently  and  stir  with  a 
rod  till  dissolved.     Test  this  solution  as  follows: 

1. — Boil  the  solution.  Absence  of  coagulation  shows  absence  of 
albumin  and  globulin. 

2. — To  a  portion  apply  the  HN03  and  heat  test  for  albumin  (p.  42). 

3. — To  another  portion  apply  the  acetic  acid  and  potassium  fer- 
rocyanide  test  for  albumin  (Exp.  10,  p.  43). 

(B).  With  the  reserved  portion  of  the  filtrate  from  tube  1  make 
the  following  tests: 

1. — Heat  a  portion  to  boiling.  What  does  the  absence  of  coagu- 
lation mean? 

2. — To  a  little  of  the  liquid  (1  c.c.)  apply  the  biuret  test  as  given 
above  under  a — 1  (p.  72). 

Exactly  neutralize  the  remainder  of  the  solution  with 
dilute  NaOH  and  test  for  albumoses  as  follows: 

3. — To  a  portion  apply  the  HNOa  and  heat  test  for  albumose 
(p.  50,  Exp.  2  6). 

4. — To  the  yellowish  liquid  obtained  in  preceding  test  (B  3)  add  an 
excess  of  NH4OH.  An  orange  yellow  color  results — the  xanthoproteic 
reaction. 

5. — To  a  portion  add  1 — 2  drops  of  dilute  acetic  acid  and  a  drop 
or  two  of  ferrocyanide  solution.  If  no  precipitate  forms  add  some 
NaCl  solution  according  to  directions  given  on  p.  50,  (Exp.  2  c). 

6. — To  another  portion  of  the  liquid  add  1—2  drops  of  fresh  tan- 
nic acid  solution.     Heavy  white  precipitate. 

3. — After  tube  2  has  been  kept  for  3 — 5  days  at  40°,  filter  the  con- 
tents. Saturate  10  c.c.  of  the  liquid  with  (NHJ2S04  according  to  the 
directions  given  above  (2  A,  p.  72).  The  liquid  is  cloudy,  but  very 
little  albumose  is  precipitated?    Why? 

Filter  the  saturated  liquid  and  to  a  portion  of  the  filtrate  apply 
the  biuret  test  as  given  above  under  a — 1  (p.  72). 

4. — The  fibrin  in  tubes  3  and  4,  in  a  few  days  at  40°,  is  completely 
dissolved  by  the  acid  present.     When  this  occurs  unite  the  contents 


74  PHYSIOLOGICAL,  CHEMISTRY. 

of  the  two  tubes  and  filter.  Exactly  neutralize  the  filtrate  according 
to  directions  given  under  Exp.  22,  p.  46.  A  heavy  white  precipitate 
shows  the  presence  of  acid  albumin,  or  as  it  is  sometimes  called  syn- 
tonin.     Pepton  may  also  form  but  will  remain  in  solution. 

IV.     Examination  of  Stomach  Contents. 

*  The  stomach  and  contents  of  a  recently  fed  rabbit  (or 
larger  animal)  are  cut  up,  diluted  with  about  500  c.c.  of 
water  and  placed  at  40°  for  about  one  hour.  The  mixture 
is  then  filtered  through  muslin.  This  dilute  gastric  juice 
is  used  for  the  following  experiments: 

I. — Test  the  reaction  with  litmus  paper.     It  is  distinctly  acid. 

2. — Test  portions  of  the  liquid  for  free  HC1  according  to  I,  1  and 

2,  (p.  68). 

3. — Test  a  portion  for  lactic  acid  with  Uffelmann's  reagent, 
(p.  70). 

4. — To  10  c.c.  of  the  solution  add  a  shred  of  fibrin  or  a  flake  of 
coagulated  egg  albumin.  Set  aside  at  40°  for  2 — 3  hours.  If  not  dis- 
solved let  the  tube  remain  at  this  temperature  over  night.  Then 
filter,  and  to  the  filtrate  apply  the  biuret  test  in  the  cold  (a  1,  p.  72). 

5. — Apply  the  biuret  test  direct  to  a  portion  of  the  dilute  gastric 
juice  and  compare  the  intensity  of  the  reaction  with  that  obtained 
in  4. 

6. — In  each  of  three  test-tubes  place  10  c.c.  of  fresh  milk.  To 
tube  1  add  2  c.c.  of  the  solution,  previously  carefully  neutralized.  To 
tube  2  add  one  drop  of  commercial  rennet  solution.  Tube  3  serves  as 
a  control.  Set  the  tubes  aside  at  40°  and  examine  occasionally  during 
the  next  hour. 

7. — Test  for  pepsin. — The  following  test  is  applicable  to  vomited 
matter,  or  to  the  liquid  obtained  from  a  stomach.  Dilute  20  c.c.  of  the 
liquid,  if  necessary,  and  filter.  To  one-half  of  the  filtrate  in  a  test- 
tube  add  a  few  shreds  of  washed  fibrin,  or  a  flake  of  coagulated  egg 
albumin.  Set  aside  at  40°  for  }4 — 1  hour.  The  fibrin  should  dissolve, 
the  egg  albumin  requires  more  time.  If  no  digestion  takes  place  it 
may  be  due  to  the  absence  of  HC1,  or  of  pepsin,  or  of  both. 

To  the  other  half  of  the  filtrate  add  an  equal  volume  of  0.5%  HCL 


GASTRIC  JUICE.  75 

This  is  prepared  by  diluting- 6  c.c.  of  concentrated  HC1  (1.19  specific 
gravity)  with  water  to  500  c.c. 

To  the  mixture  of  filtrate  and  acid  add  fibrin  or  egg  albumin  and 
set  aside  at  40°  as  above.  If  in  both  of  these  tests  the  fibrin  or 
albumin  remains  undissolved  it  is  due  to  the  absence  of  pepsin. 

Each  student  will  receive  five  "unknowns"  and  these 
are  to  be  tested  for  lactic  acid,  free  HC1,  pepsin,  and 
rennet  according  to  the  directions  given  above  under  IV 
1,  2,  3,  6,  7.  One  or  more  of  these  may  be  expected  in  such 
an  unknown.     Report  the  results. 

The  flakes  of  coagulated  egg  albumin  are  best  prepared 
by  gradually  pouring  a  dilute  solution  of  the  egg  albumin, 
with  constant  stirring,  into  boiling  water. 


CHAPTER    VI. 
PANCREATIC  SECRETION. 

The  pancreatic  secretion  is  a  clear  thick  alkaline  fluid, 
rich  in  solids  and  possesses  very  active  ferment  properties. 
It  contains  at  least  three  distinct  ferments,  besides  albu- 
min, leucin,  fats,  soap  and  salts.  These  solid  constituents 
make  up  about  10%  of  the  secretion.  After  a  pancreatic 
fistula  has  been  in  place  for  sometime  the  secretion  is 
altered.  It  becomes  thinner,  strongly  alkaline,  and  shows 
little  or  no  proteolytic  action.  The  amount  of  solids  in 
this  altered  secretion  scarcely  exceeds  two  per  cent.  The 
quantity  of  the  secretion  given  off  in  a  period  of  24  hours  is 
not  definitely  known. 

The  ingestion  of  food  stimulates  the  flow  of  the  pan- 
creatic fluid.  There  is,  therefore,  no  secretion  during-  star- 
vation and  it  is  intermittent  in  carnivorous  animals  where 
some  time  elapses  between  meals.  On  the  other  hand 
secretion  is  going  on  almost  continually  in  herbivorous 
animals  because  digestion  is  uninterruptedly  taking  place. 

As  stated  above  the  pancreatic  secretion  contains  at 
least  three  distinct  ferments  or  enzymes  splitting  up  re- 
spectively fats,  carbohydrates  and  proteids. 

The  neutral  fat  which  is  taken  into  the  body  with  the 
food  is  acted  upon  by  one  of  the  ferments,  steapsin  or  pialyn, 
and  is  split  up  by  hydration  or  saponification  into  free  fatty 
acids  and  glycerin.  This  ferment  is  very  readily  decom- 
posed by  acids  and  may  be  absent,  therefore,  from  old  pan- 
creas. Only  a  small  portion  of  the  fat,  however,  undergoes 
this  change.  The  free  acids  now  combine  with  sodium  car- 
bonate to  form  soaps  and  the  resulting  soap  solution  readily 


PANCREATIC    SECRETION.  77 

emusifies  the  remaining-  neutral  fat  and  thus  brings  it  into 
a  finely  divided  condition  suitable  for  absorption.  A  con- 
siderable portion  of  the  fat  may  at  times  be  decomposed 
into  free  fatty  acids  through  the  activity  of  bacteria.  The 
free  fatty  acids  are  not  absorbed  as  such,  but  appear  to  be 
regenerated  in  the  intestinal  walls,  by  synthesis,  into  neu- 
tral fat.  Only  a  very  small  amount  of  fat  seems  to  be  ab- 
sorbed as  soap. 

The  cleavage  of  fats  by  the  pancreatic  ferment  and  the 
subsequent  emulsification  is  necessary  to  the  proper  ab- 
sorption of  fats.  In  addition  to  the  pancreatic  secretion, 
the  bile  plays  an  important  part  in  the  absorption  of  fat. 
It  is  well  known  that  closure  of  the  bile-duct,  whether  ex- 
perimentally or  in  disease,  as  in  icterus,  is  followed  by 
diminished  absorption  and  hence  increased  excretion  of  fat, 
more  especially  fatty  acids,  in  the  feces.  Some  fat,  how- 
ever, continues  to  be  absorbed  even  in  the  absence  of  the 
bile  secretion.  The  pancreatic  secretion  is  necessary  since 
no  absorption  of  fat  takes  place  when  the  pancreas  is 
extirpated.  In  the  latter  case,  however,  milk  continues 
to  be  absorbed  owing  to  the  already  emulsified  con- 
dition of  the  fat.  Some  fat  may,  at  times,  be  absorbed 
even  after  total  extirpation  of  the  pancreas,  since  bacterial 
ferments  may  split  up  the  fat  and  thus  emulsification  and 
hence  absorption  may  result. 

The  second  ferment  of  the  pancreas  acts  on  starches, 
splitting-  up  the  bodies  into  dextrin  and  iso-maltose.  This 
ferment  is  spoken  of  as  amylolytic  or  diastatic,  and  resem- 
bles in  its  action  the  ptyalin  of  the  saliva.  It  is  probably 
not  identical  with  the  saliva  ferment.  It  is  soluble  in 
water  and  in  glycerin;  insoluble  in  alcohol.  This  diastatic 
ferment  appears  to  be  absent  during-  the  first  few  weeks  of 
infant  life.  At  the  temperature  of  the  body  it  acts  rapidly 
on  boiled  starch,  converting  this  into  amylodextrin,  ery- 
throdextrin,  achroo-dextrin,  iso-maltose  and  maltose.     By 


78  PHYSIOLOGICAL  CHEMISTRY. 

the  action  of  a  special  inverting-  ferment  the  maltose  then 
is  converted  into  glucose  in  which  form  the  carbohydrates 
are  chiefly  absorbed.  Other  mono-saccharides  as  laevulose 
and  galactose  may  also  be  absorbed  direct.  It  is  possible 
for  small  amounts  of  dextrin  and  for  milk  sugar  to  reach 
absorption. 

Sugar  is  absorbed  very  rapidly,  so  much  so,  indeed, 
that  if  a  very  large  amount  be  ingested  at  one  time  it  ap- 
pears in  the  urine.  This  condition,  known  as  alimentary 
glycosuria,  does  not  occur  when  large  quantities  of  starch 
are  ingested.  Although  the  pancreatic  gland  is  necessary 
for  the  complete  absorption  of  all  the  starch  ingested,  it  is 
a  noteworthy  fact  that  about  one  half  of  the  starch  ingested 
will  still  be  absorbed  after  total  extirpation  of  the  pan- 
creatic gland.  This  may  be  explained  by  the  diastatic 
action  possessed  by  many  bacteria. 

The  third  ferment  of  the  pancreatic  secretion  is  proteo- 
lytic in  its  action  and  is  known  as  trypsin.  This  ferment 
does  not  exist  as  such  in  the  substance  of  the  gland,  but  is 
represented  by  a  parent-substance  trypsinogen,  which  is 
most  abundant  in  the  gland  in  from  14 — 18  hours  after  a 
meal.  This  zymogen  during  the  process  of  secretion  is  con- 
verted into  the  enzyme,  trypsin.  Just  how  this  takes  place 
is  not  definitely  known.  This  conversion  can  be  accom- 
plished artificially  by  the  action  of  air,  water,  acids,  very 
weak  alkalis  and  various  other  substances.  Stronger  al- 
kalis prevent  cleavage  of  the  zymogen.  It  is  probable 
that,  as  in  the  case  of  pepsin,  the  pancreatic  secretion 
of  different   animals   contains    slightly  different  trypsins. 

Trypsin,  like  some  other  ferments,  in  its  purest  condition 
gives  proteid  reactions.  It  is  soluble  in  water,  insoluble  in 
alcohol  and  in  glycerin.  "When  in  an  impure  state,  however, 
it  may  be  dissolved  by  glycerin.  This  is  true  of  the  other 
enzymes.  In  neutral  or  slightly  alkaline  solution  it  is 
readily  destroyed  at  50°.     It  is  also  destroyed  by  gastric 


PANCREATIC    SECRETION.  79 

juice  and  unlike  pepsin  it  digests  fibrin  in  alkaline,  neutral 
or  even  very  faintly  acid  solutions.  It  is  destroyed  by 
mineral  acids,  but  not  as  a  rule  by  organic  acids.  The 
fibrin  in  tryptic  digestion  does  not  swell  and  is  not  irregu- 
larly eaten  away  as  is  the  case  in  peptic  digestion.  The 
fibrin  digestion  with  trypsin  takes  place  most  rapidly  at 
about  40°,  and  in  slightly  alkaline  solution  (0.3%  Na2CO„). 

In  view  of  the  fact  that  trypsin  acts  best  under  the 
conditions  mentioned,  it  is  evident  that  the  products  of  the 
t^-ptic  digestion  will  be  mixed  with  various  bacterial  pro- 
ducts unless  special  attention  is  given  toward  inhibiting 
the  growth  of  these  micro-organisms. 

In  actual  experiments,  therefore,  thymol  or  chloroform 
is  added  to  suppress  the  bacteria.  In  the  intestines,  of 
course,  during  pancreatic  digestion  the  bacteria  are  unhin- 
dered in  their  action.  Among  the  products  resulting  from 
the  action  of  trypsin  proper  on  fibrin  may  be  mentioned 
albumoses,  pepton,  leucin,  tyrosin,  asparaginic  acid,  lysin, 
ammonia  and  proteinochromogen.  True  pepton  is  formed 
much  more  readily  in  tryptic  than  in  peptic  digestion. 
This  pepton,  on  prolonged  digestion,  is  eventually  of  the 
kind  known  as  anti-pepton,  whereas  the  hemi-pepton  has 
been  decomposed  yielding  products  such  as  leucin,  tyrosin, 
etc.  Trypsin  dissolves  gelatin  yielding  a  gelatin-pepton. 
The  collagens  or  gelatin-yielding  connective  tissues  are 
not  acted  upon  until  they  have  been  altered  by  heat  or 
acids.     Trypsin  has  no  action  on  fats  or  carbohydrates. 

Cut  up  the  fresh  pancreatic  gland  into  very  fine  pieces,  or  better 
pass  it  through  an  Enterprise  fruit-press.  The  pulp  thus  obtained 
can  be  used  direct,  or  mixed  with  several  volumes  of  water. 

Place  about  10  c.c.  of  the  pulpy  mixture  in  a  small  beaker,  add 
2~>  c.c.  of  water  and  boil  for  about  10  minutes.  Crush  the  hard  coagu- 
lated lumps  in  a  mortar  and  return  to  the  liquid.  Reserve  this  for 
experiments  1  b,  3  b. 

1. — Cleavage  action  on  fats. — The  fat  or  oil  employed  for  this  test 
should  be  strictly  neutral.     It  can  be  obtained  in  this  condition  by  the 


80  PHYSIOLOGICAL  CHEMISTRY. 

following-  process:  Place  about  10  c.c.  of  the  oil  (cotton-seed  oil  or 
butter)  in  a  small  separatory  funnel,  add  20  c.c.  of  water  and  render 
the  mixture  distinctly  alkaline  with  NaOH.  Then  add  an  equal  vol- 
ume of  ether  and  shake  till  the  fat  dissolves.  Draw  off  the  aqueous 
liquid  and  to  the  ether  add  an  equal  volume  of  water,  and  shake  again 
to  wash  the  ether.  Remove  the  aqueous  layer  and  wash  once  more 
with  water.  Transfer  the  ether  solution,  filtered  if  need  be,  to  a 
porcelain  dish  and  allow  the  ether  to  evaporate.  The  neutral  fat  is 
left  behind. 

Place  in  each  of  two  test-tubes  about  3 — 4  c.c.  of  the  neutral  fat, 
15  c.c.  of  water  and  a  few  drops  of  concentrated  aqueous  blue  litmus 
solution. 

(a).  To  one  test-tube  add  about  5  c.c.  of  the  fresh  pancreatic 
pulp  mixture  and  shake. 

(b).  To  the  second  tube  add  one-half  of  the  liquid  containing- the 
boiled  pulp  and  shake.  If  the  contents  of  the  two  tubes  react  acid 
add,  drop  by  drop,  a  Na20O3  solution  (2%)  until  the  mixture  is  dis- 
tinctly alkaline. 

Place  the  tubes  in  an  incubator  at  40°  for  6 — 8  hours,  or  less. 
Compare  the  reaction  of  the  tubes.  Reserve  the  two  mixtures  for 
the  next  experiment.  If  the  mixtures  remain  too  long  at  this  temper- 
ature bacteria  develop  give  rise  to  acids,  and  reduce  the  litmus.  The 
two  tubes  will  then  be  quite  alike  and  will  give  the  same  results  in 
the  next  experiment. 

Under  the  influence  of  a  ferment  in  the  pancreas,  known 
as  steapsin,  or  pialyn,  the  neutral  fat  is,  in  part,  decom- 
posed into  free  fatty  acid  and  glycerin. 

2. — Emulsifying  action  on  fats. — After  digesting  the  two  mixtures 
at  40°  for  6 — 8  hours  in  the  preceding  experiment,  shake  thoroughly 
and  take  y3 — }4  of  the  contents  of  each  tube  and  treat  as  follows: 

(a).  To  a  portion  of  the  mixture  from  Exp.  1  a  add  about  1  c.c. 
of  Na2COs  solution  (2%)  and  shake  thoroughly.  The  liquid  becomes 
milky  and  on  standing  the  fat  does  not  rise  to  the  surface.  Examine 
a  drop  of  the  emulsion  under  the  microscope. 

(6).  To  a  portion  of  the  mixture  from  Exp.  1  b  add  Na2C03  solu- 
tion as  above  and  shake.  The  liquid  does  not  emulsify.  The  fat  rises 
rapidly  to  the  surface  on  standing.  If  bacterial  decomposition  has 
taken  place,  or  if  the  fat  was  not  neutral  in  the  beginning,  some 
emulsification  will  result. 

Why  is  the  fat  emulsified  in  one  case  and  not  in  the  other? 


PANCREATIC    SECRETION.  81 

3. — Diastatic  action. — Prepare  a  starch  paste  by  boiling-  1  g.  of 
powdered  starch  with  100  c.c.  of  water.  Place  in  each  of  two  test- 
tubes  15  c.c.  of  the  starch  paste. 

(a).  To  one  add  about  2  c.c.  of  the  pancreatic  pulp  mixture,  mix 
and  place  in  a  water-bath  at  40°. 

(ft).  To  the  second  add  %  of  the  boiled  pulp  mixture  and  like- 
wise set  aside  in  the  water-bath  at  40°. 

At  intervals  of  about  15  minutes  take  out  a  portion,  about  2  c.c, 
from  each  of  the  two  tubes.  Test  one-half  of  each  portion  with 
iodine  for  starch  and  dextrin.  Test  the  remainder  for  sugar  by  boil- 
ing with  Fehling's  solution.  How  soon  does  dextrin  make  its  appear- 
ance? How  early  can  sugar  be  recognized?  When  is  the  achromic 
point  reached? 

Note  the  change  in  the  appearance  of  the  two  tubes  and  the 
difference  in  results.  Compare  this  action  of  pancreas  with  that  of 
saliva. 

4. — Proteolytic  action. — In  a  50  c.c.  Erlenmeyer  flask,  provided  with 
a  cork,  place  about  5  g.  of  fresh  fibrin  and  about  20  c.c.  of  chloroform 
water.  This  is  prepared  by  adding  2  c.c.  of  chloroform  to  50  c.c.  of 
water  and  shaking  thoroughly.  To  the  fibrin  and  chloroform  water 
add  about  5  c.c.  of  the  pancreatic  pulp  and  mix  well.  Render  the 
mixture  distinctly  alkaline  by  the  addition  of  a  few  drops  of  Na2C03 
solution  (2% ).     Cork  the  flask  and  set  aside  at  40°  for  2 — 3  days. 

Occasionally  examine  the  contents  of  the  flask  and  compare  the 
rate  of  digestion  with  that  of  gastric  juice.  Note  that  the  fibrin  does 
not  swell  up  as  in  gastric  digestion,  and  that  the  edges  are  evenly 
eaten  away. 

The  contents  of  the  flask  are  finally  slightly  acidulated  with 
acetic  acid,  boiled  and  filtered.  The  filtrate  may  contain  albumose, 
pepton,  tyrosin  and  leucin. 

a).     To  a  portion  of  the  filtrate  apply  the  biuret  test  in  the  cold. 

(&).  To  another  portion  add  a  few  drops  of  bromine  water  and 
shake.  A  pink  to  a  purple-red  color  (proteinochrom)  develops.  This 
is  known  as  the  bromint  reaction  and  is  due  to  an  unknown  substance, 
proteinochromogen  or  tryptophan. 

This  substance  in  its  composition  resembles  haemato- 
porphyrin  and  bilirubin,  and  is  also  related  to  the  animal 
pigments  as  melanin.  It  would  seem  as  if  this  substance 
was  utilized  to  build  up  haemoglobin  and  other  pigments. 


82  PHYSIOLOGICAL  CHEMISTRY. 

(c).  Concentrate  the  remainder  of  the  filtrate  on  a  watch-glass 
to  a  small  volume  (a  few  c.c.)  and  set  aside  in  a  cool  place  over  night. 
Examine  the  deposit  with  the  microscope  for  tyrosin  which  forms 
characteristic  bundles  of  needles,  and  for  leucin  balls.  If  no  deposit 
forms  concentrate  to  a  thin  syrup  and  again  set  aside  for  exam- 
ination. 

Leucin  .  C6H]3N02. 

This  compound  was  formerly  considered  to  be  a-amido- 
caproic  acid,  but  the  more  recent  studies  of  Schulze  and 
Likiernik  have  shown  it  to  be  a-amido  isobutyl-acetic  acid, 
(CH3)2CH  .  CH2  .  CH(NH2)  .  C02H.  Leucin  is  readily  formed 
and  is  a  constant  cleavage  product  in  the  decomposition  of 
proteids,  gelatin  and  horn.  This  decomposition  occurs  in 
pancreatic  digestion ;  may  be  brought  about  by  the  action 
of  acids  and  alkalis  at  high  temperatures;  and  may  also 
occur  as  a  temporary  bacterial  product  during  putrefaction. 
Plant  proteids,  as  well  as  animal  proteids,  can  give  rise  to 
leucin.  It  can  be  readily  prepared  from  proteids,  or  better 
white  horn,  by  boiling  with  dilute  HC1.  Leucin  has  been 
found,  in  diseased  conditions,  in  various  organs  and  glands 
of  the  body,  in  pus,  blood,  and  in  decomposed  epidermis 
such  as  is  found  on  the  feet  and  between  the  toes.  In  the 
latter  case  the  peculiar  odor  is  largely  due  to  decomposition 
products  of  leucin.  This  compound  occurs  with  tyrosin, 
in  the  urine  in  liver  diseases,  especially  in  acute  yellow 
atrophy.     They  are  not  present  in  normal  urine. 

Leucin  is  dextro-rotatory  in  acid  or  in  alkaline  solu- 
tions, but  in  neutral  solutions  is  inactive.  On  heating  with 
baryta  it  becomes  inactive.  By  the  action  of  Penicillium 
the  inactive  form  is  changed  into  a  laevo-rotatory  variety. 
Several  isomeric  leucins  have  been  prepared  synthetically. 

In  the  pure  condition  leucin  forms  glistening  white 
plates,  which  do  not  readily  moisten  when  touched  with 
water.  As  usually  met  with,  however,  it  forms  balls  or 
aggregations  of  spherical  bodies  which  often  show  light 
radial  markings  and  are  faintly  refractive  to  light.     When 


PANCREATIC    SECRETION.  83 

impure  leucin  is  more  readily  soluble  than  when  pure.  It 
is  readily  soluble  in  water  (27  parts);  more  readily  in  hot 
water.  It  is  difficulty  soluble  in  alcohol,  but  is  readily 
soluble  in  acids  and  alkalis.  It  forms  salts  with  acids  and 
bases. 

The  following-  ex}3eriments  are  made  with  leucin  fur- 
nished by  the  laboratory: 

1. — To  a  drop  of  water  on  a  slide  add  a  little  leucin,  about  the 
size  of  a  pin-head.  Observe  the  behavior  on  contact  with  water. 
Then  mix,  place  on  a  cover-glass  and  examine  under  the  microscope. 
Sketch  the  crystals  observed. 

Then  add  a  drop  of  water  to  the  edge  of  the  cover-glass  and 
gently  heat  over  a  flame  until  the  leucin  dissolves.  Set  aside  to  cool 
slowly,  then  examine  and  sketch  the  crystals. 

2. — To  a  few  c.c.  of  urine  in  a  watch-glass  add  a  little  leucin,  mix 
and  heat  till  it  dissolves.  Concentrate  on  the  water-bath  to  a  small 
volume;  cover  with  a  beaker  and  set  aside  over  night.  Then  place 
the  watch-glass  under  the  microscope  and  examine  with  a  low  power. 
Typical  light  yellow  spherules  or  mulberry-like  masses  of  leucin  will 
be  found.  Transfer  some  of  the  deposit  to  a  slide  and  examine  with 
a  higher  objective. 

3. — To  about  1  c.c.  of  water  in  a  test-tube  add  leucin  and  shake 
till  it  ceases  to  dissolve.     Divide  into  two  portions: 

(a).     To  one  add  a  drop  of  dilute  HC1. 

(&).     To  the  other  add  a  drop  of  dilute  NH4OH. 

4. — Place  some  leucin  in  a  glass  tube,  about  six  inches  long,  and 
open  at  both  ends,  and  heat  gently  in  an  inclined  position.  A  portion 
of  the  leucin  is  sublimed  as  a  woolly  deposit.  At  the  same  time  an 
odor  of  amylamin  is  given  off. 

5. — Dissolve  some  leucin  in  a  little  water  and  render  the  solution 
alkaline  with  NaOH.  Then  add  1 — 2  drops  of  dilute  0uSO4  solution. 
The  cupric  hydrate  precipitate  which  forms  at  first,  redissolves,  since 
with  leucin  it  forms  a  soluble  compound.  The  solution  is  colored  blue, 
and  on  heating  does  not  reduce.  This  action  of  leucin  is  similar  to 
that  of  glycocoll,  of  tartrates,  and  of  bile. 

6. — Place  in  a  dry  test-tube  a  piece  of  solid  KOH  about  one-half 
an  inch  long:  add  some  leucin  and  a  drop  of  water.     Heat  cautiously 


84  PHYSIOLOGICAL  CHEMISTRY. 

till  the  KOH  melts.  Place  a  strip  of  moist  red  litmus  paper  near  the 
mouth  of  the  tube.  What  is  the  result  and  to  what  is  it  due'?  Pour 
the  melted  KOH  into  a  small  beaker,  rinse  the  tube  with  a  little 
water,  and  add  this  to  the  beaker.  Place  the  beaker  in  cold  water 
and  add  very  cautiously,  drop  by  drop,  H2S04  till  the  solution  is  acid. 
Then  heat  the  beaker  over  a  flame  and  note  the  peculiar  odor  of 
valerianic  acid.  If  the  odor  is  not  marked  pour  the  liquid  into  a  test 
tube,  cork  and  set  aside  for  a  day  or  two.  On  opening  the  tube  the 
odor  will  be  perceptible. 

7. — Place  some  crystals  of  leucin  on  a  platinum  foil,  add  a  drop 
or  two  of  nitric  acid  (1.20  specific  gravity)  and  evaporate  carefully  to 
dryness.  A  colorless  scarcely  visible  residue  remains.  Xow  add  a 
few  drops  of  NaOH  and  warm.  The  residue  dissolves  forming  a  clear 
or  slightly  colored  solution.  On  cautious  concentration  an  oily  drop 
remains  which  does  not  moisten  the  foil  but  rolls  about  readily.  This 
is  known  as  Scherer's  test  and  is  verj-  characteristic. 

For  the  detection  of  leucin  in  urine  see  p.  86. 


Tyrosin,  C9HnN03. 

Tyrosin  has  been  prepared  synthetically  and  is  there- 
fore  known  to  be   para-oxyphemTl-a-amidopropionic  acid, 

OH 

A 
I  I 
V 

CH2  .  CH(NtL)  .  C02H. 

Tyrosin  is  a  constant  cleavage  product  resulting-  from 
the  action  of  trypsin,  bacteria,  acids,  or  alkalis  on  animal 
or  vegetable  proteids  or  horn.  It  is  not  obtained  from 
gelatin  or  gelatin-yielding  tissues.  It  is  as  a  rule  accom- 
panied by  leucin.  It  is  present  in  old  cheese  and  its  name 
refers  to  this  source.  It  is  present  in  the  intestines  during 
proteid  digestion,  but  is  not  present  in  the  tissues,  blood  or 
urine  of  the  normal  body.  It  is  met  with  in  the  urine  in 
phosphorous  poisoning  and  in  acute  yellow  atrophy  of  the 
liver. 


PANCREATIC    SECRETION.  85 

It  forms  delicate  colorless  silky  needles  which  melt  at 
235°.  The  crystals  often  group  in  bundles  and  when  very 
impure  may  form  leucin-like  balls.  It  is  very  difficulty 
soluble  in  cold  water  (1 — 2,400),  more  soluble  in  hot  water, 
insoluble  in  alcohol  or  ether.  It  is  readily  soluble  in  dilute 
alkalis  and  in  dilute  mineral  acids.  In  acid  solution  tyro- 
sin  is  laevo-rotatory,  whereas  the  synthetic  product  or  that 
prepared  by  the  aid  of  alkalis  is  dextro-rotatory. 

The  tyrosin  necessary  for  the  following-  experiments  is 
furnished  by  the  laboratory: 

1. — Treat  a  small  portion  in  the  same  way  as  in  Exp.  1,  under 
Leucin.  How  can  the  bundles  of  fine  needles  of  tyrosin  be  distin- 
guished from  similar  bundles  of  needles  of  fatty  acids?  of  calcium 
sulphate? 

2. — Test  the  solubility  of  tyrosin  according;  to  the  directions 
given  in  Exp.  3,  under  Leucin. 

3. — To  some  water  in  a  test-tube  add  a  little  tyrosin,  then  a  few 
drops  of  Millon's  reagent.  Heat  the  liquid  till  it  begins  to  boil.  It 
colors  rose-red,  and  on  standing  becomes  dark-red  and  may  yield  a 
red  precipitate.  This  is  known  as  Hofmanris  reaction  and  is  due  to 
the  presence  of  the  oxy-phenyl  group  in  tyrosin.  What  other  sub- 
stances give  this  reaction  with  Millon's  reagent? 

4. — Place  some  tyrosin  in  a  dry  test-tube,  add  a  few  drops  of  con- 
centrated H2S04.  Place  the  test-tube  in  a  water-bath  and  heat  at 
100'  for  about  half  an  hour.  Then  cool  and  pour  the  contents  into  a 
small  beaker  containing  some  water.  To  this  liquid  now  add  BaC03 
in  small  portions  while  stirring,  until  the  reaction  ceases  to  be  acid. 
Filter  the  liquid  and  concentrate  the  nitrate  to  a  very  small  volume. 
To  this  concentrated  liquid  add  a  drop  or  two  of  very  dilute  FeCl3. 
A  beautiful  violet  color  develops.      This  test  is  known  as  Piria's 

miction. 

5. — Place  some  crystals  of  tyrosin  on  a  platinum  foil,  add  nitric 
acid  (1.2  sp.  g.)  and  warm.  The  tyrosin  becomes  bright  orange  yellow 
and  dissolves.  Evaporate  very  cautiously  to  dryness  when  a  deep 
yellow,  transparent  residue  remains.  Add  a  few  drops  of  NaOH  and 
a  deep  reddish-yellow  solution  results.  This  on  evaporation  leaves  an 
intense  blackish-brown  residue  (Sch< n  r's  test). 


86  PHYSIOLOGICAL  CHEMISTRY. 

A  similar  reaction  is  given  by  other  substances  and  consequently 
it  is  not  characteristic. 

6. — To  a  boiling"  aqueous  solution  of  tyrosin  add  some  one  per 
cent,  acetic  acid  and  then  sodium  nitrite  solution,  drop  by  drop.  A 
beautiful  red  color  develops  (Wurster). 

7. — To  a  hot  aqueous  solution  of  tyrosin  add  some  dry  quinone. 
The  liquid  becomes  colored  a  ruby-red  (Wurster). 

*  Detection  of  Leucin  and  Tyrosin  in  Urine. 

Tyrosin  because  of  its  difficult  solubility  may  occur  in 
the  sediment  in  urine,  but  if  only  a  small  amount  is  present 
it  may  be  in  solution.  Inasmuch  as  leucin  is  more  soluble 
it  will  be,  as  a  rule,  in  solution  in  the  urine. 

Precipitate  the  urine  with  basic  acetate  of  lead,  filter 
and  remove  the  lead  from  the  filtrate  by  hydrogen  sulphide. 
Then  concentrate  the  solution  as  low  as  possible  and  set 
aside  to  crystallize.  Examine  under  the  microscope  for 
crystals  of  leucin  and  tyrosin.  If  leucin  is  present  it  can 
be  removed  by  means  of  warm  alcohol. 


CHAPTER     VII. 
BILE. 

Bile  is  a  mixture  of  the  secretion  of  liver  cells  and  of 
mucin  derived  from  the  cells  lining-  the  bile-bladder  and 
duct.  It  is  a  thick,  tenacious  fluid  and  is  alkaline  in  reac- 
tion. The  specific  gravity  ranges  from  1.01  to  1.04.  The 
color  of  bile  varies  in  different  animals.  It  may  be  light 
5*ellow,  brownish  yellow,  brownish  green,  green,  and  green- 
ish blue.  Human  bile  is  yellowish,  at  times  greenish.  Bile 
j^ossesses  a  pronounced  bitter  taste.  It  does  not  coagulate 
on  heating.  Human  bile  contains  true  mucin,  whereas,  ox. 
bile  contains  but  traces  of  mucin  and  instead  a  nucleo- 
albumin. 

The  quantity  of  bile  secreted  in  21  hours  is  subject  to 
considerable  variation  even  in  health.  In  the  case  of 
fistula;,  from  0.6  to  1  liter  of  bile  has  been  observed  to  be 
secreted  in  21  hours,  but  the  secretion  under  these  condi- 
tions can  hardly  be  considered  as  normal  bile.  The  actual 
quantity  given  off  in  a  day  is  probably  not  less  than  half 
a  liter.  After  a  proteid  diet  the  secretion  is  increased, 
whereas,  with  fats  and  carbohydrates  it  is  less  marked. 
The  secretion  is  also  decreased  in  starvation.  The  secre- 
tion is  continuous  but  with  variable  intensity.  Inasmuch 
as  the  bile  flows  from  the  bladder  under  very  little  pressure, 
a  slight  obstruction  in  the  duct  may  lead  to  retention  of 
the  bile.  As  a  result  the  bile  constituents  are  absorbed 
and  may  appear  in  the  urine.  Human  bile  as  it  is  found  in 
the  bladder  after  death  has  been  found  to  contain  from 
7 — 18  per  cent,  of  solids.  The  bile  as  it  flows  from  the 
liver  in  a  fistula,  contains  much  less  solids,  1 — 4  per  cent. 


88  PHYSIOLOGICAL  CHEMISTRY. 

The  bile,  therefore,  becomes  concentrated  in  the  bladder 
by  absorption  of  water. 

Bile  contains  as  characteristic  constituents  certain 
salts  of  bile  acids,  bile  pigments,  and  small  quantities  of 
lecithin,  cholesterin,  soap,  neutral  fat,  urea,  and  salts  of 
calcium,  magnesium,  iron,  and  copper. 

The  bile  acids  are  usually  present  as  sodium  salts.  In 
some  sea-fish  they  are  in  combination  with  potassium.  It 
is  customary  to  speak  of  two  bile  acids,  glycocholic  and 
taurocholic.  The  former  on  cleavage  yields  glycocoll  and 
cholic  acid;  the  latter  taurin  and  cholic  acid.  Inasmuch  as 
this  cholic  acid  is  but  one  of  several  cholic  acids  known,  it 
follows  that  there  is  a  group  of  glycocholic  acids  and  a 
group  of  taurocholic  acids.  Human  bile  yields  three  bile 
acids.  The  bile  of  some  animals  may  contain  only  glyco- 
cholic acid,  or  only  taurocholic  acid;  whereas  in  some, 
variable  mixtures  of  the  two  acids  are  present.  Thus,  the 
taurocholic  acid  predominates  in  the  bile  of  carnivorous 
animals,  birds,  reptiles,  and  fish.  The  bile  from  the  rabbit 
and  the  hog  contains  almost  entirely  glycocholic  acid. 
That  from  herbivorous  animals  as  a  rule  contains  vari- 
able quantities  of  both.  Both  glycocoll  and  taurin  are 
amido  acids.  Taurin  contains  S  as  a  characteristic  consti- 
tuent. According  to  Hammarsten  the  bile  of  some  ani- 
mals contains  a  third  group  of  bile  acids  which  are  rich  in 
S,  and  which  in  their  behavior  to  mineral  acids  resemble 
ethereal  sulphates. 

The  bile-acid  salts  are  precipitated  from  their  solution 
in  alcohol,  on  the  addition  of  ether,  as  fine  needles.  The 
bile  acids  and  their  salts  are  dextro-rotatory. 

A  large  number  of  pile  pigments  are  known,  but  in  nor- 
mal bile,  as  a  rule,  there  are  but  two,  bilirubin  and  biliver- 
din.  The  former  can  be  obtained  as  a  reddish  yellow, 
the   latter   as   a   greenish  powder.      The  color   of  bile  is 


BILE.  89 

due  to  the  preponderance  of  one  or  the  other  of  these  two 
pigments.  Ox-bile  has  both  pigments.  The  other  bile  pig- 
ments, as  bilifuscin,  biliprasin  and  bilicyanin,  have  been 
isolated  from  bile  stones  and  altered  bile. 

The  bile  pigments  are  soluble  in  alkalis,  insoluble  in 
acids,  and  yield  insoluble  compounds  Avith  calcium  and 
other  metals.  Bilirubin  is  slightly  soluble  in  alcohol  and 
in  ether,  readily  soluble  in  chloroform.  Biliverdin  is  insol- 
uble in  chloroform.  Bilirubin,  in  addition  to  being  in  the 
bile,  is  met  with  in  bile  stones  as  a  calcium  compound;  in 
old  blood  extravasations  (haematoidin);  and  in  urine  and 
tissues  during  icterus. 

The  source  of  bilirubin  is  undoubtedly  hasmatin.  On 
reduction  it  yields  hydrobilirubin  which  is  closely  related, 
if  not  identical,  with  stercobilin  (found  in  the  intestines) 
and  with  urobilin  of  urine.  On  oxidation  it  yields  biliver- 
din. The  amount  of  pigment  in  the  bile  is  usually  only  a 
few  hundredths  of  a  per  cent.,  rarely  0.1  per  cent. 

As  to  the  origin  of  these  bile  constituents  it  may  be 
said  that  the  bile  acids  are  elaborated  by  the  cells  of  the 
liver,  not  elsewhere  in  the  body.  The  bile  pigments,  with- 
out doubt,  can  be  formed  in  other  parts  of  the  body  than 
in  the  liver,  but  under  normal  conditions  the  liver  is  the 
organ  where  they  are  formed.  Taurin  and  glycocoll  result 
from  the  decomposition  of  proteids  in  any  part  of  the  body. 

1. — Place  some  dilute  bile  (1 — 5)  in  a  test-tube  and  heat  to  boil- 
ing. Immerse  a  strip  of  red  litmus  paper,  then  remove  and  wash 
with  water.     The  reaction  is  distinctly  alkaline. 

2. — Place  about  5  c.c.  of  bile  in  a  test-tube,  add  10  c.c.  of  water, 
mix  and  filter  if  necessary.  To  the  clear  liquid  add  acetic  acid.  A 
cloudiness  or  distinct  precipitate  of  mucin  or  nucleo-albumin  forms  on 
standing.     This  is  not  marked  in  ox-bile. 

3. — Filter  the  cloudy  liquid  obtained  in  Exp.  2,  and  apply  the 
biuret  test  to  the  clear  filtrate.  Absence  of  proteids.  Notice  also, 
that  the  Cu(OH)2  precipitate  which  forms  redissolves  in  the  bile  solu- 
tion and  yields  a  blue  liquid  which  on  heating  gives  a  black  precipi- 


90  PHYSIOLOGICAL,  CHEMISTRY. 

tate.     What  is  the  cause  of  this  black  precipitate?    What  other  sub- 
stances redissolve  Cu(OH)2  and  yield  blue  solutions'? 

4.— To  about  20  c.c.  of  bile  in  an  evaporating  dish  add  about  5  g. 
of  animal  charcoal  and  evaporate  on  the  water-bath,  with  frequent 
stirring,  to  complete  dryness.  Transfer  the  residue  to  a  150  c.c. 
Erlenmeyer  flask,  provided  with  a  cork  and  condensing-  tube,  add 
about  30  c.c.  of  absolute  alcohol  and  boil  on  the  water-bath  for  about 
half  an  hour.  Cool  and  filter  into  a  dry  flask  (or  a  50  c.c.  test-tube  on 
foot).  To  the  alcoholic  filtrate  add  anhydrous  ether  till  a  permanent 
precipitate  forms.  Then  cork  and  set  aside  in  a  cool  place  over 
night.  The  sodium  salts  of  glycocholic  and  taurocholic  acids  crystal- 
lize out.  Filter  off  the  crystalline  deposit  and  save  the  filtrate. 
Squeeze  the  crystals  as  dry  as  possible  on  the  filter  in  the  funnel, 
then  dry  between  several  sheets  of  filter  paper.  Save  the  crystals 
for  subsequent  tests. 

5. — The  alcohol-ether  filtrate  from  the  preceding  experiment 
contains,  among  other  things,  cholesterin.  Place  this  filtrate  in  an 
evaporating  dish  and  allow  the  ether  to  evaporate  spontaneously, 
then  cautiously  evaporate  to  dryness  on  the  water-bath.  Rub  up  the 
residue  thoroughly,  with  some  ether,  filter  the  ethereal  solution  into 
a  small  beaker  or  watch-glass,  and  allow  the  ether  to  evaporate  spon- 
taneously. Examine  the  residue  under  the  microscope  for  the  char- 
acteristic crystals  of  cholesterin.  Fatty  crystals,  in  the  form  of 
needles,  are  likely  to  be  present. 

6. — Detection  of  bile  acids.—  On  the  side  table  are  two  sets  of  bile 
solutions— bile  dissolved  in  water,  and  bile  dissolved  in  urine.  These 
solutions  are  made  up  in  the  following  strengths:  1 — 10,  1 — 100,  1 — 500, 
1 — 1,000.  Apply  the  following  tests  first  to  the  "bile-water"  dilu- 
tions, then  to  corresponding  dilutions  of  bile  with  urine.  Tabulate 
the  results. 

Place  about  5  c.c.  of  each  of  these  solutions  in  test-tubes  and 
apply  the  following  test,  noting  carefully  the  delicacy  of  the  reaction. 

(a).  To  the  liquid  to  be  tested  add  about  two-thirds  its  volume 
of  concentrated  sulphuric  acid.  The  acid  is  allowed  to  run  down  the 
side  of  the  tube  slowly,  so  as  not  to  mix.  The  temperature  should  not 
rise  over  60 — 10°.  If  necessary,  therefore,  cool  partly  under  the 
hydrant,  then  add  2—3  drops  of  a  solution  of  cane-sugar  (1—10)  and 
tap  the  tube  gently.  A  pink  to  a  red  or  violet  color  develops  accord- 
ing to  the  amount  of  bile  acids  present.  The  foam  which  forms  on 
shaking  is  likewise  colored  pink.     This  is  known  as  Pettenkofer's  test, 


BILE.  91 

and  depends  upon  the  formation  of  furfurol.  An  excess  of  sugar  and 
too  much  heat  must  be  carefully  avoided.  Observe  the  difference  in 
the  delicacy  of  the  reactions  in  aqueous  and  urine  solutions  of  bile. 
Oleic  acid  gives  a  somewhat  similar  reaction. 

To  some  water  in  a  test-tube  add  sulphuric  acid  as  above,  then 
about  five  drops  of  the  sugar  solution.  Notice  the  yellow  to  dark- 
brown  color  that  forms.  Repeat  this  blank  test  with  urine,  acid,  and 
five  drops  of  the  sugar  solution. 

(&).  Furfurol  test. — Since  Pettenkofer's  test  depends  upon  the 
formation  of  furfurol,  out  of  the  sugar  added,  it  can  therefore  be 
made  with  a  furfurol  solution. 

To  a  few  c.c.  of  the  solution  to  be  tested  add  one  drop  of  a  1.0% 
aqueous  furfurol  solution,  then  add  slowly  as  in  the  preceding  test 
about  an  equal  volume  of  concentrated  sulphuric  acid,  cool  somewhat 
if  necessary,  and  avoid  an  excess  of  furfurol.  The  reaction  is  often 
less  intense  than  in  6  «. 

Apply  this  test  to  some  diluted  bile.  Dissolve  a  little  of  the 
crystallized  bile  acids  obtained  in  Exp.  4,  in  some  water.  Observe  the 
foaming  of  the  liquid  when  shaken.  Divide  this  solution  into  two 
portions  and  test  one  according  -to  Pettenkof  er;  the  other  with 
furfurol. 

*(c).  Detection  of  bile  acids  in  the  urine  (Hojjpe-Sey lev's 
Method). — The  test  given  above  under  6  a  is  usually  em- 
ployed. It  should  be  remembered,  however,  that  sub- 
stances may  be  present  in  urine  which  will  give  a  reaction 
similar  to  that  of  bile  acids.  Moreover,  in  highly  colored 
urines  the  reaction  can  be  readily  masked.  In  such  cases 
the  following  method  of  Hoppe-Seyler,  though  somewhat 
long,  will  give  good  results.  About  100  c.c.  of  the  urine  is 
evaporated  to  a  syrup  and  the  residue  extracted  with 
strong  alcohol.  The  alcoholic  filtrate  is  evaporated  to 
dryness,  and  the  residue  obtained  is  dissolved  in  water. 
The  aqueous  solution  is  precipitated  with  lead  acetate  and 
ammonia.  The  precipitate  is  washed,  then  transferred  to 
an  evaporating  dish  or  flask  and  extracted  with  boiling 
alcohol.  The  alcoholic  solution  is  filtered  while  hot.  A 
few  drops  of  soda  solution  are  added  to  the  filtrate  and  this 
is  then  evaporated  to  dryness. 


92  PHYSIOLOGICAL  CHEMISTRY. 

The  dry  residue  can  now  be  dissolved  in  water,  the 
solution  slightly  acidulated  with  H2S04  and  filtered.  The 
aqueous  filtrate  can  now  be  tested  directly  for  bile  acids 
according-  to  6  a  or  b. 

7. — Detection  of  bile  pigments. — On  the  side-table  will  be  found  five 
bottles  containing  urine  diluted  with  bile  in  the  following"  propor- 
tions: 1—10,  1—20,  1—50,  1—100,  1—500.  Apply  the  following-  tests  to 
these  solutions  and  tabulate  the  results. 

(a).  Gmelin's  test. — Place  some  bile  or  the  suspected  urine  in  a 
small  evaporating  dish  and  add  a  drop  or  two  of  fuming  HN03 — a  play 
of  colors,  green,  blue  to  violet  results.  With  ox-bile  the  colors  are 
weak  and  rapidly  change.  In  urine  the  green  color  is  especially  im- 
portant since  indican  may  also  give  a  blue  color.  Various  modifica- 
tions of  this  test  have  been  suggested  and  of  these  the  following  are 
especially  useful. 

{a  1).  Filter  the  bile  solution  or  suspected  urine.  Then  add  a 
drop  or  two  of  fuming  nitric  acid  to  the  moist  filter  paper.  The  col- 
ored rings  are  very  distinct.  This  test  (Bosenbach's)  is  much  more 
satisfactory  than  the  preceding  and  is  especially  useful  when  the 
urine  is  highly  colored. 

(a  2).  To  a  few  c.c.  of  fuming  HN03  in  a  test-tube  add  slowly 
some  dilute  bile  solution  or  the  suspected  urine,  so  that  the  two 
liquids  do  not  mix.  Colors  develop  at  the  zone  of  contact.  Finally 
mix  the  contents;  a  decided  green  color  forms,  especially  on  standing. 

(&).  Huppert's  reaction. — To  about  10  c.c.  of  the  diluted  bile  or 
suspected  urine  add  a  little  calcium  chloride,  then  an  excess  of  am- 
monium or  sodium  carbonate.  The  bilirubin-calcium  compound  is 
precipitated.  Filter,  wash  the  precipitate,  then  transfer  while  moist 
to  a  test-tube  and  fill  it  half  full  of  alcohol  which  has  been  acidulated 
with  sulphuric  acid.  Immerse  the  tube  for  10 — 15  minutes  in  a  water- 
bath,  heated  so  that  the  contents  of  the  tube  are  kept  near  the  boil- 
ing point.  The  solution  becomes  colored  an  emerald  to  a  bluish  green. 
Now  cool  the  contents  of  the  tube,  then  add  fuming  HN03.  The  green 
color  changes  to  blue,  violet,  and  red.  This  test  is  very  delicate  and 
is  especially  useful  when  the  urine  is  highly  colored,  or  contains  much 
indican  or  blood  pigments. 

(c).  Iodine  test. — Place  the  diluted  bile  or  suspected  urine  in  a 
test-tube,  incline  the  tube  and  add  cautiously  2 — 3  c.c.  of  a  dilute 
tincture  of  iodine  so  that  it  forms  a  layer.     Immediately  or  after  a 


BILE.  93 

few  minutes  a  bright  green  ring  forms  at  the  zone  of  contact  [JEtosirv- 
Smith).  This  reaction  is  almost  as  delicate  as  that  of  Huppert. 
After  ingestion  of  antipyrin  the  urine  will  give  a  similar  green  ring 
with  iodine. 

*(d).  Jones'  test. — This  is  claimed  to  be  the  most  deli- 
cate test  for  bile  pigments.  Place  50  c.c.  of  the  suspected 
urine  or  a  mixture  of  bile  and  urine  (1 — 500)  in  a  glass  stop- 
pered C3Tlinder,  add  a  few  drops  of  10%  HC1,  then  BaCl2  in 
excess  and  5  c.c.  of  chloroform,  and  shake  vigorously  for 
several  minutes.  Set  aside  for  about  ten  minutes  for  the 
precipitate  and  chloroform  to  settle.  Transfer  the  chloro- 
form and  precipitate  by  means  of  a  pipette  to  a  test-tube. 
Immerse  the  tube  in  a  water-bath  having  a  temperature  of 
about  80°.  The  chloroform  evaporates  in  about  10  minutes. 
Remove  the  test-tube  and  after  a  few  minutes  when  the 
precipitate  has  settled  decant  the  supernatant  liquid.  The 
precipitate  is  colored  yellow,  if  bile  pigment  is  present. 
Allow  three  drops  of  concentrated  HN03  (to  which  about  yi 
volume  of  fuming  HN03  has  been  added)  to  run  down  the 
side  of  the  tube.     The  characteristic  play  of  colors  develops. 

(e).  Acidulate  some  dilute  bile  with  acetic  acid,  add  a  few  c.c. 
of  chloroform  and  shake.  The  chloroform  dissolves  the  bilirubin  and 
is  colored  yellow.  Icteric  urine  treated  in  this  manner  likewise 
colors  the  chloroform. 

Bile  Stones. 

The  calculi  found  at  times  in  the  gall-bladder  of  man 
consist  chiefly  of  cholesterin.  They  may  be  grayish  or  yel- 
lowish white,  wax-like  in  appearance,  or  may  be  colored 
from  a  light  red  to  a  dark  brown.  The  color  depends  upon 
the  amount  of  bilirubin  present.  This  pigment  is  not  free 
but  in  combination  with  calcium.  The  number  of  stones 
present  in  the  gall-bladder  may  vary  from  a  few  to  several 
hundred.  The  size  will,  therefore,  vary  considerably,  from 
that  of  a  grain  of  wheat  to  stones  from  x/z — 1  inch  in  diame- 


94  PHYSIOLOGICAL  CHEMISTRY. 

ter.  As  a  result  of  friction  the  stones  frequently  show 
smooth  triangular  faces.  The  larger  stones  when  cut  in 
two  and  polished  show  generally  a  concentric  arrangement. 
When  they  consist  of  pure  cholesterin  the  stones  will  float 
on  water.     Small  amounts  of  fat  may  also  be  present. 

The  bile-stones  as  usually  found  in  the  gall-bladder  of 
cattle  consist  largely  or  wholly  of  the  calcium-bilirubin 
compound.  Similar  calculi  are  met  with  occasionally  in 
man.  These  pigment  stones  may  contain  metals  such  as 
iron  and  copper,  and  even  at  times  zinc  and  manganese. 
Unlike  the  cholesterin  stones  they  are  always  heavier  than 
water. 

A  third  form  of  bile  stones  very  rarely  found  in  man 
consists  chiefly  of  calcium  carbonate  and  phosphate. 

Examination  of  bile-stones. — Pulverize  a  sm  all  bile-stone  and  place 
the  powder  in  a  test-tube.  Add  a  mixture  of  alcohol  and  ether,  equal 
parts,  and  warm  gently  until  the  powder  ceases  to  dissolve.  Decant 
the  ether-alcoholic  solution  into  a  watch-glass  or  evaporating  dish 
and  allow  it  to  evaporate  spontaneously.  If  the  crystals  are  imper- 
fect redissolve  in  hot  alcohol  and  allow  the  solution  again  to  evapor- 
ate spontaneously. 

Save  the  crystals  for  the  subsequent  tests  for  cholesterin. 

If  there  is  a  residue  insoluble  in  the  ether-alcoholic  mixture  add 
to  it  some  dilute  HC1.  An  effervesence  indicates  a  carbonate  (CaC03). 
If  an  insoluble  residue  still  remains  wash  it  with  water  and  examine 
for  bile  pigments. 

Evaporate  the  HC1  solution  to  dryness  and  ignite;  then  dissolve 
the  residue  in  dilute  HC1  and  add  NH4OH.  A  blue  color  indicates  the 
presence  of  copper. 

Cholesterin,  C27H45OH. 

This  is  a  common  constituent,  though  in  minute 
quantity,  of  the  normal  fluids  and  tissues  of  the  body. 
It  is  very  abundant  in  nervous  tissue,  especially  in  the 
white  matter.  Under  pathological  conditions  it  is  met 
with    especially    in    bile -stones.       It    is    also    present    in 


BILE.  95 

atheroma  nodules,  in  tubercular  masses,  tumors,  sputum, 
pus  transsudates  and  cystic  fluids.  It  is  rarely  present  in 
the  urine  and  then  in  small  amount.  A  rare  urinary  choles- 
terin  calculus  has  been  reported  by  Horbaczewski.  Com- 
pounds closely  resembling-  cholesterin,  possibly  isomers, 
are  found  in  plants  (phytosterins)  and  by  some  these  have 
been  regarded,  though  incorrectly,  as  the  source  of  choles- 
terin in  animals.  The  cholesterin  present  in  bile  passes 
into  the  intestines  and  a  small  portion  may  be  excreted  as 
such.  Most  of  it,  however,  is  reduced  to  a  hydro-compound 
— stercorin  or  coprosterin. 

Cholesterin  forms  white,  glistening  crystals  which 
under  the  microscope  appear  as  very  thin  transparent 
plates  with  a  more  or  less  notched  corner.  The  crystals 
melt  at  145°  whereas  plant  cholesterin  melts  at  133°.  It  is 
insoluble  in  water,  in  dilute  acids,  and  in  alkalis.  It  is 
readily  soluble  in  boiling  alcohol  from  which  on  cooling  it 
recrystallizes.    It  is  readily  soluble  in  ether  and  chloroform. 

1. — Examine  under  the  microscope  and  sketch  the  character- 
istic crystals  of  cholesterin  obtained  from  a  bile-stone. 

2. — To  some  crystals,  on  a  slide  under  the  microscope,  add  a  drop 
of  dilute  H2S04  (five  parts  of  acid  to  one  part  of  water).  The  edges 
of  the  crystals  show  a  bright  carmine-red  color  which  changes  to 
violet. 

3. — To  some  crystals  as  in  Exp.  2,  add  a  drop  of  dilute  H2S04,  then 
a  drop  of  iodine  solution.  The  crystals  turn  gradually  violet,  bluish- 
green,  then  blue. 

4. — Dissolve  a  few  crystals  in  a  little  chloroform  in  a  dry  test- 
tube,  then  add  an  equal  volume  of  sulphuric  acid  and  shake.  The 
chloroform  becomes  blood-red,  then  cherry-red  and  purple.  The  acid 
liquid  shows  a  green  fluoresence  (Salkowski).  The  color  of  the  chloro- 
form is  quickly  discharged  if  it  is  poured  into  a  moist  test-tube. 

5. — Dissolve  some  cholesterin  in  2  c.c.  of  chloroform,  add  10  drops 
of  acetic  anhydride,  and  then  drop  by  drop,  concentrated  H2SO^. 
The  mixture  becomes  red,  blue,  and  finally  green  [IAebermarm's 
Cholest(  rol  reaction). 


96  PHYSIOLOGICAL  CHEMISTRY. 

6.— To  a  little  cholesterin  in  an  evaporating"  dish  add  a  few  drops 
of  HC1,  and  a  drop  of  very  dilute  FeCl3.  On  evaporating  to  dryness  a 
blue  color  results. 

7. — Place  a  little  of  the  dry  cholesterin  in  a  dry  test-tube,  add 
2 — 3  drops  of  propionic  anhydride  and  carefully  heat  over  a  small 
flame  till  melted.  On  gradually  cooling  the  mass  becomes  violet, 
then  green,  blue  and  red. 

*  Detection  of  cholesterin  in  urine. — Inasmuch  as  choles- 
terin is  lighter  than  water  it  will  be  found  when  present  in 
urine,  floating-  on  the  surface  as  a  thin  pellicle.  A 
microscopic  examination  will  often  decide  the  nature  of 
this  film.  This  is  also  true  of  transsudates  and  other  path- 
ological fluids  where  the  crystals  are  often  well  formed. 
Some  crystals  may  be  dragged  mechanically  to  the  bottom. 
In  the  absence  of  typical  crystals  it  will  be  necessary  to 
employ  the  following  method: 

Extract  the  urine  with  ether  which  takes  up  fat  and 
cholesterin.  Remove  the  ethereal  layer  and  allow  it  to 
evaporate  spontaneously.  Examine  the  residue  under  the 
microscope  for  the  characteristic  crystals  of  cholesterin. 
If  there  is  any  doubt  owing  to  the  presence  of  fats  these 
must  be  removed  by  saponification.  For  this  purpose  dis- 
solve the  residue  in  hot  alcohol,  add  some  strong  alcoholic 
solution  of  potassium  hydrate  and  heat  on  the  water-bath 
for  some  time.  Finally  evaporate  to  dryness,  and  extract 
the  residue  of  soaps  with  ether.  This  ethereal  solution  on 
evaporation  will  now  give  a  residue  free  from  fat. 


CHAPTER    VIII 


BLOOD. 


Blood  is  usually  a  dark-red,  thick,  opaque  fluid.  The 
average  specific  gravity  of  normal  blood  is  about  1.058,  and 
depends  primarily  upon  the  amount  of  haemoglobin  present. 
It  consists  of  blood  corpuscles  (red  and  white)  and  blood- 
jDlates  suspended  in  the  liquid  portion — the  plasma.  The 
solid  blood  corpuscles  in  man  ma3^  constitute  nearly  one-half 
the  weight  of  the  blood.  In  some  animals  as  the  ox,  they  make 
up  but  one-third  of  the  weight  of  the  blood.  The  blood  of 
adult  man  contains  per  cubic  millimeter  about  5,000,000  red, 
and  7,500  white  blood  cells  and  about  250,000  blood  plates. 
The  blood  of  women  contains  about  4,500,000  red  cells  per 
cubic  millimeter. 

The  blood  possesses  a  distinct  alkaline  reaction,  due 
chiefly  to  sodium  carbonate.  The  alkalinity  is  decreased 
considerably  in  febrile  conditions,  diabetic  coma,  cancer,  and 
after  excessive  muscular  exercise.  This  decrease  is  due  to 
the  increased  production  of  acids,  such  as  sulphuric,  phos- 
phoric, and  volatile  fatty  acids,  which  result  from  the  in- 
creased disintegration  of  protein  tissues. 

The  red  blood  corpuscles  of  man  and  mammals  are  round, 
bi-concave  discs  which  contain  no  nucleus.  The  llama, 
camel  and  related  species  constitute  an  exception  to 
the  latter  statement,  since  their  blood  in  common  with 
that  of  birds,  amphibians,  fish  and  reptiles,  contains 
nucleated  red  blood  cells  which  are  bi-convex  and  more  or 
less  elliptical.  The  size  of  the  red  blood  cell  varies  some- 
what among  the  different  species  of  mammals,  but  not  suffi- 
ciently  to  enable  one  to   distinguish  human  from  animal 


98  PHYSIOLOGICAL  CHEMISTRY. 

blood.  This  is  especially  true  in  the  legal  examination  of 
blood  stains.  The  presence  of  blood  cells  and  the  produc- 
tion of  hasmin  crystals  justifies  the  assertion  that  the  stain 
is  due  to  blood,  merely  this  and  nothing-  more.  The  size 
of  the  cells  and  absence  of  nuclei  may  exclude  the  blood  of 
certain  animals,  but  it  does  not  prove  that  it  is  human  blood. 

The  average  diameter  of  the  red  blood  cells  in  the 
blood  of  man  is  7.5  p.  or  about  msta  of  an  inch.  The  blood 
cells  of  the  ox  and  horse  are  about  tsW;  sheep,  about  sinus; 
goat,  about  <nrW.  The  blood  corpuscles  of  but  very  few 
mammals  are  larger  than  those  in  human  blood;  as  a  rule 
they  are  smaller. 

The  opacity  of  the  blood  is  due  to  the  suspended  blood 
corpuscles,  just  as  that  of  milk  is  due  to  the  suspended  fat 
globules.  The  cells  consist  of  a  stroma  or  shell  which  holds 
in  its  meshes  the  haemoglobin.  If  the  stroma  is  dissolved 
or  altered  so  as  to  allow  the  haemoglobin  to  go  into  solution 
the  blood  then  becomes  transparent  or  "  laky." 

The  white  blood  corpuscles,  leucocytes  or  lymphoid 
cells,  differ  considerably  in  form  and  size.  They  are  larger 
and  lighter  than  the  red  blood  cells  and  contain  1 — 4  nuclei. 
They  show  amaeboid  motion.  A  marked  increase  in  the 
number  of  white  blood  cells  (leucocytosis)  is  observed  in 
leukamia.  The  leucocytes  consist  largely  of  the  complex 
proteid,  nucleo-histon.  After  a  rich  proteid  diet  the  blood 
may  contain  an  appreciable  amount  of  albumose,  or  so- 
called  pepton.  This  albumose,  however,  is  not  in  solution 
in  the  plasma  but  is  contained  within  the  leucocytes.  "When 
these  undergo  disintegration  as  in  the  case  of  large  ab- 
scesses, the  albumose  is  set  free,  and  when  this  is  now 
absorbed  by  the  blood  it  is  promptly  excreted  by  the  kid- 
neys and  appears  in  the  urine.  If  albumose  (or  haemoglo- 
bin) is  injected  directly  into  the  blood  it  is  excreted  at  once 
in  the  urine.  If  serum  albumin  is  injected  into  the  blood  it 
is  not  excreted  by  the  kidneys,  but  egg- albumin  would  be 
excreted. 


BLOOD.  99 

The  blood  plates  are  supposed  by  some  to  be  derived 
from  nuclei  and  hence  consist  chiefly  of  nuclein. 

The  plasma  contains  about  8.2  per  cent,  of  solids.  Of 
this  amount  6.9  per  cent,  is  due  to  proteins  and  about  0.87 
per  cent,  due  to  inorganic  constituents,  such  as  chlorides, 
phosphates,  and  carbonates. 

There  are  three  albuminous  substances  contained  in 
the  plasma,  namely:  fibrinogen,  serum  globulin  and  serum 
albumin. 

Fibrinogen  is  a  most  important  constituent  of  plasma. 
It  is,  moreover,  present  in  chyle,  lymph,  exsudates  and 
transsudates.  In  general  it  resembles  the  globulins,  but  is 
distinguished  from  serum  globulin  especially  by  its  beha- 
vior to  NaCl,  which  precipitates  it  on  semi-saturation. 
Fibrinogen  solutions  (usually  about  0.5  per  cent.)  coagulate 
when  heated  to  56°  or  less. 

The  globulins,  fibrinogen  and  serum  globulin,  make  up 
most  of  the  proteins  of  the  blood.  The  relative  amounts  of 
globulin  and  albumin  vary  in  different  species  and  even  in 
the  same  species  of  animal.  Serum-  or  para-globulin  is 
unchanged  by  the  clotting  of  blood.  It  can  be  precipitated 
by  a  current  of  CO.,,  or  by  saturation  with  MgS04.  It  coa- 
gulates at  75°. 

Serum  albumin  is  present  in  plasma,  serum,  lymph, 
exsudates  and  transsudates.  Pathologicall}T  it  may  ap- 
pear, accompanied  by  globulin,  in  the  urine.  It  coagulates 
usually  at  about  70 — 75°,  but  if  the  solution  is  concentrated 
and  little  or  no  NaCl  is  present  it  may  coagulate  at  50°. 
Serum  albumin,  and  possibly  the  other  proteins  of  the 
blood,  is  made  by  the  epithelial  cells  of  the  intestine  out  of 
the  pepton  prepared  by  the  digestive  fluids.  The  pepton 
that  is  made  in  the  stomach  and  in  the  intestine  is  not 
absorbed  and  carried  through  the  body  as  such,  but  is 
regenerated,  synthesized,  to  serum  albumin  by  the  cells  of 
the  intestinal  wall.  The  blood  coming  from  the  intestines 
does  not  contain  pepton  or  albumose  in  solution. 


100  PHYSIOLOGICAL,  CHEMISTRY. 

Coagulation  of  blood. — Within  a  few  minutes  after  blood 
is  taken  from  an  animal  it  clots,  forming  a  solid  jelly. 
This  clot  is  essentially  a  net-work  of  fibrin  threads  con- 
taining- in  its  meshes  the  blood  corpuscles  and  the  fluid 
part  of  the  blood.  Eventually  the  clot  shrinks  and  a  light 
yellow  fluid  (blood-serum)  is  squeezed  out.  If  the  blood  as 
soon  as  it  is  drawn  from  the  animal  is  rapidly  stirred  with 
the  hand,  or  whipped  with  a  bundle  of  sticks,  glass-rods,  or 
wire,  the  solid  clot  will  not  form,  but  instead  the  hand  or 
stirring  rods  will  be  covered  with  shreds  of  fibrin,  or  as  it' 
is  sometimes  called  "blood  fibre."  The  resulting  fluid  is 
spoken  of  as  " defibrinated  blood";  it  is  blood  serum  con- 
taining in  suspension  blood  corpuscles.  The  fibrin  shreds 
when  thoroughly  washed,  are  pure  white,  and  in  many 
respects  resemble  the  coagulated  white  of  an  egg. 

Coagulation  of  the  blood  implies,  therefore,  the  forma- 
tion of  fibrin.  This  change  according  to  Schmidt  is  brought 
about  by  the  action  of  the  fibrin  ferment  derived  from  leu- 
cocytes, on  serum  globulin  and  fibrinogen.  Subsequently, 
Hammarsten  showed  that  the  serum  globulin  was  not  essen- 
tial to  clotting.  That  is  to  say,  the  fibrin  ferment  acting 
on  fibrinogen  yields  fibrin. 

When  the  blood  is  taken  from  an  animal  and  received 
in  a  flask  containing  some  potassium  oxalate  it  will  not 
clot.  The  absence  of  coagulation  in  this  case  is  due  to  the 
precipitation  of  calcium  as  an  oxalate.  In  other  words, 
calcium  is  essential  to  the  formation  of  fibrin  (Arthus  and 
Pages).  The  role  of  calcium  in  coagulation  has  not  been 
clearly  explained  until  recently. 

The  fibrin  ferment  is  apparently  a  globulin,  not  a 
nuclein.  It  is  not  present  in  fresh  arterial  blood  (Jako- 
wicki);  nor  is  it  present  in  pepton  or  histon  plasma  (Lilien- 
feld);  nor  is  it  present  in  oxalate  plasma  (Hammarsten, 
Pekelharing).  The  absence  .of  fibrin  ferment  in  the  circu- 
lating blood  has  led  Lilienfeld  to  believe  that  it  was  not 
the  cause  but   rather  the   product   of   coagulation.     This, 


BLOOD.  101 

however,  is  not  correct,  for  while  it  is  true  that  the  fibrin 
ferment  does  not  exist  ready  made  in  the  blood  the  parent 
substance  of  the  fibrin  ferment  is  present.  This  zymogen 
has  been  designated  as  prothrombin.  It  yields  the  fibrin 
ferment  when  calcium  is  present.  Calcium,  therefore,  is 
necessary  to  the  formation  of  the  fibrin  ferment  and  not  to 
the  fibrin  proper.  This  is  seen  in  the  fact  that  a  fibrin  fer- 
ment, free  from  calcium,  on  contact  with  a  fibrinogen  solu- 
tion, likewise  free  from  calcium,  yields  at  once  a  typical 
coagulum  of  fibrin.  In  order  to  demonstrate  this  relation 
of  calcium  to  fibrin  formation  it  is  not  necessary  to  prepare 
pure  fibrin  ferment  and  pure  fibrinogen.  If  ordinary  blood- 
serum  is  treated  with  potassium  oxalate,  to  remove  cal- 
cium, and  this  serum  which  contains  fibrin  ferment  is  added 
to  oxalate  plasma,  hence  likewise  free  from  calcium,  a 
typical  fibrin  clot  will  result. 

The  present  view  regarding  the  clotting  of  blood  may 
be  summarized  as  follows: 

1. — Calcium  salts  +  Prothrombin  (zymogen)  =  Fibrin-ferment. 
2. — Fibrin-ferment  -f-  Fibrinogen  =  Fibrin. 

LilienfekVs  belief  that  fibrinogen  on  contact  with 
nuclein  and  other  compounds  yields  a  cleavage  product, 
thrombosin,  which  with  calcium  yields  fibrin,  is  incorrect. 
In  the  first  place  true  fibrin  contains  mere  traces  of  cal- 
cium, and  secondly,  his  thrombosin  has  been  shown  to  be 
fibrinogen  which  forms  an  insoluble  calcium  compound  in 
slightly  alkaline  fluids,  poor  in  salts.  It  is  important  to 
note  in  this  connection  that  calcium  is  likewise  essential  to 
the  coagulation  of  milk.  The  casein  of  the  milk  is  changed 
by  rennet  to  a  modified  form  (paracasein)  which  unites  with 
calcium  to  form  the  curd  or  cheese  (see  Exp.  8  under  milk). 


102  PHYSIOLOGICAL  CHEMISTRY 

Defibrinated  Blood. 

I.  Microscopic  Examination. 

1. — Examine  a  drop  of  fresh  blood  under  the  microscope.  Meas- 
ure the  diameter  and  sketch  the  red  and  white  blood  cells.  What  is 
the  difference  between  the  blood  cells  of  mammals  and  of  birds,  rep- 
tiles, etc. 

2. — Dilute  some  fresh  blood  with  water  and  examine  as  before. 
Observe  and  sketch  the  crenated  blood  cells. 

II.  Spectroscopic  Examination. 

1. — Add  1  c.c.  of  defibrinated  blood  to  50  c.c.  of  distilled  water 
and  shake  thoroughly.  Place  some  of  the  dilute  blood  in  a  test-tube 
and  suspend  this  about  an  inch  before  the  slit  of  the  spectroscope 
(Position  No.  1).  The  test-tube  should  not  be  more  than  one-half  inch 
in  diameter.  A  fish-tail  burner  placed  about  three  inches  from  the 
slit  serves  as  a  source  of  light. 

Observe  the  two  absorption  bands  of  oxy-hcemoglobin  and  their 
position  on  the  scale  in  the  spectrum. 

Place  in  the  flame  a  platinum  wire  previously  dipped  in  a  solu- 
tion of  sodmm  chloride.  Notice  the  characteristic  yellow  line  of 
sodium,  its  position  on  the  scale,  and  its  relation  to  the  two  absorption 
bands. 

2. — Now  swing  into  position  the  little  outside  prism  of  glass  so 
that  it  shuts  off  the  lower  half  of  the  slit.  Place  a  light  about  three 
inches  in  front  of  the  left  face  of  this  prism.  The  spectrum  of  haemo- 
globin appears  in  the  lower  half  of  the  field  of  view  while  superposed 
above  it  is  a  clear  spectrum.  Place  a  tube  of  the  blood,  diluted  and 
well  shaken  as  above,  between  this  second  light  and  the  left  face  of 
the  prism  (Position  No.  2).  The  spectrum  from  this  tube  is  now 
thrown  above  that  from  the  tube  in  front  of  the  slit.  The  two 
spectra  of  oxy-haamoglobin  coincide. 

(a).  To  tube  No.  1  before  the  slit,  add  1 — 2  drops  of  freshly  pre- 
pared ammoniacal  ferro-tartrate  solution  (Stokes'  solution),  and  ex- 
amine at  once.  The  two  bands  of  oxy-haemoglobin  soon  disappear, 
giving  place  to  the  single  wide  band  of  reduced  haemoglobin.  Compare 
this  spectrum  with  the  superposed  one  of  oxy-haemoglobin.  Note  the 
change  in  the  color  of  the  blood. 

The  Stokes'  solution  is  prepared  as  follows:  Dissolve  2  parts  of 


BLOOD.  103 

ferrous  sulphate  and  3  parts  of  tartaric  acid  in  water,  then  render 
alkaline  by  addition  of  NH4OH. 

(&).  To  tube  No.  2,  the  one  on  the  left,  now  add  5 — 6  drops  of 
strong  ammonium  sulphide  and  examine.  In  a  few  minutes  the  single 
band  of  reduced  haemoglobin  takes  the  place  of  the  two  bands  of  oxy- 
hemoglobin.    The  spectra  of  the  two  tubes  now  coincide. 

3. — To  the  tube  of  reduced  haemoglobin  in  position  No.  1,  obtained 
in  Exp.  2  a,  add  a  few  drops  of  concentrated  NaOH.  The  single  ab- 
sorption band  becomes  replaced  by  two  bands,  resembling  those  of 
oxy-haemoglobin,  but  shifted  a  little  to  the  right.  The  left  band  is 
the  darker  of  the  two.  On  standing  a  few  minutes  the  spectrum 
increases  in  intensity  so  that  the  two  bands  merge  together;  in  that 
case  dilute  with  an  equal  volume  of  water,  and  examine  again. 

This  spectrum  is  due  to  hcemochromogen,  or  reduced  hcematin.  This 
test  should  be  resorted  to  when  the  spectrum  of  haemoglobin  is 
doubtful. 

Compare  this  spectrum  with  the  superposed  spectrum  of  reduced 
haemoglobin  (2  &).  Then  substitute  for  the  latter  a  tube  of  the 
diluted,  well  shaken  blood,  thus  placing  the  spectrum  of  oxy-haemo- 
globin  above  that  of  haemochromogen. 

■i. — Dilute  some  defibrinated  blood  with  about  15  parts  of  water 
and  shake  well.  Place  some  of  this  solution  in  a  test-tube,  in  posi- 
tion 1.  Superpose  the  spectrum  of  oxy-haemoglobin,  using  dilute 
blood  (1 — 50)  as  in  Exp.  2.  The  upper  spectrum  of  the  very  dilute 
blood  shows  the  two  bands  of  oxy-haemoglobin,  whereas  the  lower 
spectrum,  that  of  tube  No.  1  which  contains  a  strong  solution,  is 
entirely  dark  to  the  right  of  the  sodium  line. 

To  the  tube  in  front  of  the  slit,  position  1,  now  add  1 — 2  drops  of 
a  fresh,  concentrated  solution  of  potassium  ferricyanide.  The  color 
of  the  liquid  changes  to  a  brown  and  the  spectrum  of  methcemoglobin 
appears.  An  intense  dark  band  in  the  red  with  two  less  dark  bands 
to  the  right.  If  the  liquid  is  too  concentrated  dilute  with  % — ^ 
its  volume  of  water. 

To  the  solution  of  methaemoglobin  add  a  few  drops  of  (NH4)2S2. 
The  color  and  spectrum  of  oxy-haemoglobin  reappear,  and  in  a  short 
time  give  way  to  that  of  reduced  haemoglobin  (2  b). 

5. — To  about  10  c.c.  of  concentrated  H2S04  in  a  test-tube  add 
about  five  drops  of  blood.  Shake  thoroughly  after  the  addition  of 
each  drop  of  blood  and  keep  the  contents  of  the  tube  cool.  Note  the 
dark  wine-red  color  of  the  solution. 


104  PHYSIOLOGICAL  CHEMISTRY. 

Dilute  a  portion  of  this  liquid  with  2—3  parts  of  water,  cool  and 
examine  before  the  spectroscope  (position  1)  for  the  spectrum  of 
hcematoporpliyrin.  Superpose  as  in  Exp.  2,  the  spectrum  of  oxy-haemo- 
globin  (1 — 50)  for  comparison.  Haematoporphyrin  shows  a  dark  nar- 
row band  to  the  left  and  a  wider,  darker  band  to  the  right  of  the  left 
band  of  oxy-haemoglobin-. 

Haematoporphyrin,  C16H18N203,  is  derived  from  haematin 
by  the  splitting  off  of  iron.  It  results  also  from  the  action 
of  HBr  on  haematin.  It  is  an  isomer  of  bilirubin  and  has 
been  met  with  in  urine. 

6. — To  5  c.c.  of  diluted  blood  (1 — 15)  add  2  c.c.  of  concentrated 
NaOH.  The  color  changes  to  a  cherry-red.  Now  heat  the  tube  till 
the  color  changes  to  a  brownish-green.  Examine  before  the  spectro- 
scope, position  1,  for  the  spectrum  of  alkaline  hce.rn.atin.  If  necessary 
dilute  the  contents  of  the  tube  X — Vi  with  water.  Alkaline  haematin 
shows  a  dark  band  through  the  middle  of  which  passes  the  sodium 
line.  Superpose  the  spectrum  of  oxy-haamoglobin  and  compare  the 
two  spectra.  Then  convert  the  upper  spectrum  into  reduced  haemo- 
globin as  in  Exp.  2  a,  and  again  compare. 

7. — Pass  a  current  of  illuminating  gas  for  a  few  minutes  through 
some  diluted  blood  (1 — 50). 

(a).  Place  a  tube  containing  some  of  the  blood  thus  treated 
before  the  spectroscope  in  position  1.  Superpose  the  spectrum  of 
oxy-haemoglobin  (1 — 50).  The  lower  spectrum,  due  to  carbon  monoxide 
hcemoglobin,  is  nearly  the  same  as  that  of  oxy-haemoglobin.  The  two 
bands,  however,  are  darker  and  are  removed  a  trifle  to  the  right,  so 
that  the  two  spectra  are  not  exactly  continuous.  Compare  the  color 
of  the  two  tubes. 

(6).  Now  add  to  each  tube  1 — 2  drops  of  Stokes'  solution.  Care- 
fully note  the  change  in  color  of  the  two  solutions  and  also  the  change 
in  the  spectra. 

(c).  Again  superpose  the  spectrum  of  oxy-haemoglobin  above 
that  of  CO-haemoglobin.  Then  add  to  each  tube  5 — 6  drops  of  strong 
(NH4)2S.2.    Examine  at  once,  and  after  the  lapse  of  about  five  minutes. 

(d).  Again  superpose  the  spectrum  of  oxy-haemoglobin  above 
that  of  CO-haemoglobin.  Then  add  to  each  tube  one  drop  of  a  freshly 
prepared  strong  solution  of  potassium  ferricyanide.  Examine  at 
once.     The  oxy-haemoglobin  spectrum  changes   in  a  few  seconds  to 


BLOOD.  105 

that  of  methaemoglobin,  whereas  the  spectrum  of  CO-ha^moglobin 
persists  and  is  changed  only  after  the  lapse  of  several  minutes. 
Owing-  to  the  dilution  the  spectrum  of  methaemoglobin  will  be  faint. 

CO-haemoglobin  is  a  much  more  stable  compound  than 
oxy-haemoglobin  and  for  that  reason  the  color  and  the  spec- 
trum of  the  solution  in  experiments  b,  c,  and  d,  will  change 
slowly,  if  at  all,  whereas  oxy- haemoglobin  is  readily  changed 
to  reduced  haemoglobin  in  experiments  b  and  c,  and  to  met- 
haemoglobin  in  experiment  d. 

ILL      General  Reactions. 

1. — Test  the  reaction  of  some  fresh  defibrinated  blood. 

(a).  Dip  a  moist  red  litmus  paper  for  a  few  seconds  into  the 
blood,  then  wash  at  once  in  water. 

(&).  Place  a  drop  of  aqueous  red  litmus  solution  on  a  porous  por- 
celain plate.  When  this  has  been  absorbed  apply  a  drop  of  blood  to 
the  spot  and  allow  this  to  remain  for  about  a  minute.  Then  wash  off 
with  water.  Owing-  to  the  coloring  matter  in  the  blood  this  method 
of  testing  is  much  more  delicate  than  the  preceding. 

2. — To  some  water  in  a  test-tube  add  a  drop  or  two  of  blood  and 
mix.  Then  add  tincture  of  guajac  till  the  liquid  becomes  cloudy,  and 
finally  add  some  old  oil  of  turpentine.  A  blue  color  develops  at  the 
zone  of  contact  of  the  liquids  and  is  due  to  the  oxidation  of  the 
guajac.  The  reaction  fails  with  fresh  oil  of  turpentine  owing  to  the 
absence  of  ozone. 

fa).  This  test  may  be  applied  to  urine,  suspected  of  containing 
blood,  in  the  following  manner:  Place  in  a  test-tube  equal  volumes  of 
guajac  and  old  oil  of  turpentine.  The  mixture  must  not  be  blue. 
Now  add  the  urine  cautiously  so  that  it  forms  a  layer.  If  blood  is 
present  a  bluish-green  ring  will  form  at  the  zone  of  contact.  This  is 
known  as  Almen's  Guajac  Test.  The  urine,  if  alkaline,  should  be  neu- 
tralized or  rendered  faintly  acid.  Pus  may  give  the  test  with  guajac 
alone. 

3.— To  2  c.c.  of  fresh  blood  in  a  test-tube  add,  without  shaking, 
2— .j  c.c.  of  hydrogen  peroxide.  Oxygen  is  liberated  abundantly  and 
the  liquid  foams:  the  haemoglobin  is  gradually  decomposed.  This  is 
due  to  a  so-called  catalytic  action. 


106  PHYSIOLOGICAL,  CHEMISTRY. 

4. — To  some  fresh  diluted  blood  (1 — 5)  in  a  test-tube  add  ether 
and  gently  agitate.  The  liquid  becomes  transparent  because  of  the 
solution  of  blood  cells — laky  blood. 

5. — Pass  a  current  of  illuminating  gas  for  a  few  minutes  through 
some  dilute  blood  (1 — 50).  Notice  the  cherry-red  color  of  the  solution. 
As  shown  above  in  Exp.  7  (p.  104),  CO-haemoglobin  is  a  much  more 
stable  compound  than  oxy-haemoglobin.  The  following  tests  still 
further  serve  to  demonstrate  this  fact,  and  are  of  great  value  in  dis- 
tinguishing between  the  two  forms  of  haemoglobin.  The  tests  c  and 
d  are  especially  adapted  for  the  detection  of  small  amounts  of  CO- 
haemoglobin  in  blood. 

(a).  In  one  test-tube  place  some  dilute  blood  (1 — 50);  in  another 
some  of  the  CO-haemoglobin  solution  (1 — 50).  To  each  of  these  solu- 
tions add  half  a  volume  of  strong  NaOH  solution  (1.34  specific  gravity). 
The  pure  blood  solution  becomes  brownish  (due  to  haematin),  whereas 
the  CO-haemoglobin  solution  is  unaltered  and  retains  its  cherry-red  or 
pink-red  color  (Hoppe-Seyler's  test). 

(&).  Place  5  c.c.  of  the  diluted  blood  (1 — 50)  in  a  test-tube.  In 
another  tube  place  5  c.c.  of  the  CO-haemoglobin  solution  (1 — 50).  To 
each  tube  add  an  equal  volume  of  fresh,  saturated  H2S-water  and 
shake.  The  pure  blood  changes  to  a  green,  due  to  the  formation  of 
sulphur-methaemoglobin,  whereas  the  color  of  the  CO-haemoglobin  is 
unchanged  or  fades  slowly. 

(c).  In  one  test-tube  place  5  c.c.  of  the  dilute  blood  (1 — 50);  in 
another  tube  5  c.c.  of  the  CO-haemoglobin  solution.  To  each  of  the 
tubes  add  1 — 2  drops  of  dilute  acetic  acid,  then  one  drop  of  potassium 
ferrocyanide  solution  (1 — 5).  The  proteids  in  both  solutions  are  pre- 
cipitated, but  the  precipitate  in  the  tube  of  pure  blood  is  brownish  in 
color,  whereas  that  in  the  CO-haemoglobin  tube  is  pink.  On  standing 
a  while  the  pink  color  changes  and  both  precipitates  are  then  alike. 

(cZ).  In  one  test-tube  place  5  c.c.  of  the  dilute  blood  (1 — 50);  in 
another  5  c.c.  of  CO-haemoglobin  solution  (1 — 50).  To  each  of  these 
tubes  add  an  equal  volume  of  freshly  prepared  1  per  cent,  solution  of 
tannic  acid.  The  proteids  are  again  precipitated.  The  precipitate  in 
the  tube  containing  pure  blood  is  colored  a  grayish  brown,  whereas 
that  in  the  CO-haemoglobin  tube  is  pink.  An  excess  of  tannic  acid 
may  dissolve  the  precipitate  and  should  therefore  be  avoided. 

Make  a  mixture  of  1  c.c.  of  CO-haemoglobin  solution  (1 — 50)  and 
4  c.c.  of  oxy-haemoglobin  solution  (1 — 50),  add  an  equal  volume  of  the 
tannic  acid  solution  and  compare  with  the  two  tubes  obtained  above. 


BLOOD.  107 

Haemoglobin  is  readily  decomposed  on  heating  with 
acids  or  alkalis  into  globulin  and  a  pigment.  If  oxy-haemo- 
globin  is  acted  upon  the  pigment  that  results  is  hcematin, 
whereas  with  reduced  haemoglobin  the  product  is  haemo- 
chromogen.  The  latter  decomposition  has  been  studied  in 
Exp.  3  (p.  103),  whereas  the  formation  of  ha;matin  has 
been  observed  in  Exp.  6  (p.  L04),  and  in  Exp.  ~>  a  (p.  106). 
Hamiatin  combines  with  HC1  to  form  haemin.  When  ha;min 
crystals  are  dissolved  in  alkali  ha;matin  is  formed. 

6.— Preparation  ofhcemin  crystals. — Place  in  a  small  Erlenmeyer 
na.sk  (about  30  c.c.  capacity)  provided  with  a  cork  and  condensing 
tube,  10  c.c.  of  glacial  acetic  acid  and  heat  to  boiling  on  the  water- 
bath.  Then  add,  gradually  and  with  constant  stirring,  'i  c.c.  of 
defibrinated  blood.  Continue  heating  on  the  water-bath  for  half  an 
hour.  Transfer  to  a  small  narrow  beaker  or  test-tube  and  set  aside 
over  night.  Examine  the  crystalline  deposit  microscopically  and 
sketch  the  form  of  the  ha;min  crystals. 

To  preserve  the  specimen  decant  the  acetic  acid;  then  add  10—20 
C.i  .  of  water,  stir  thoroughly  and  place  aside  to  settle.  De<  ant  off 
the  water  and  wash  in  a  similar  manner  with  alcohol;  then  stir  up 
the  crystals  with  ether  and  transfer  to  a  small  filter.  Press  the 
crystals  between  filter  paper  till  dry,  then  transfer  to  a  specimen 
tube.  The  operation  of  washing  can  be  greatly  simplified  by  the  use 
of  a  centrifugal  apparatus. 

The  recognition  of  haemin  crystals  is  of  the  greatest 
importance  in  the  identification  of  blood  stains.  Each  stu- 
dent will  receive  a  piece  of  fabric  and  a  piece  of  wood 
stained  with  blood,  or  with  some  red  dye.  These  are  exam- 
ined  in  the  following  manner  and  a  report  is  to  be  made 
upon  the  nature  of  the  stain: 

[a  .  Scrape  a  little  of  the  stain  off  the  piece  of  wood.  Place  the 
scrapings  on  a  glass  slide,  add  a  drop  of  1  per  cent,  solution  of  NaCl  and 
warm  gently  over  a  very  small  flame,  avoiding  ebullition,  until  the 
water  is  nearly  driven  off.  Then,  while  still  moist  add  1 — 2  drops 
of  glacial  acetic  acid,  cover  with  a  cover-glass  and  again  warm 
gently  over  a  small  flame  till  most  of  the  acetic  acid  has  evaporated. 


108  PHYSIOLOGICAL  CHEMISTRY. 

When  cool,  examine    under  the  microscope   for  the  characteristic 
light  brown  haemin  prisms.     Sketch  the  form  of  the  perfect  crystals. 

[b).  Soak  the  cloth  in  a  1  per  cent,  solution  of  NaCl  in  a  watch- 
glass  and  squeeze  out  the  coloring  matter  as  thoroughly  as  possible. 
Concentrate  the  liquid,  if  it  is  but  weakly  colored,  on  the  water-bath 
to  a  small  volume.  Then  place  1 — 2  drops  of  the  liquid  on  a  glass  slide, 
warm  gently,  as  above  under  a,  until  the  liquid  is  nearly  evaporated, 
then  add  1 — 2  drops  of  glacial  acetic  acid,  cover  with  a  glass-slip  and 
again  heat  till  most  of  the  acetic  acid  has  evaporated.  Cool  and 
examine  for  haemin  crystals. 

7. — The  formation  of  haematin  and  of  haemin  crystals  may  be 
utilized  for  the  detection  of  a  small  amount  of  blood  or  blood  pigment 
in  the  urine.  To  the  suspected  urine  add  NaOH  and  boil.  The  earthy 
phosphates  are  precipitated  and  are  colored  brownish-red  by  the 
haematin  (Heller's  test).  If  there  is  doubt  as  to  the  nature  of  the  color- 
ing matter  in  the  precipitate,  this  can  be  filtered  off  and  subjected  to 
the  haemin  test  according  to  the  directions  given  above  under  6  a. 

The  urine  may  be  precipitated  with  tannic  acid  and  the  pre- 
cipitate can  then  be  treated  for  haemin  crystals  as  above. 

8. — Place  about  20  c.c.  of  defibrinated  blood  in  a  beaker  and  add 
about  200  c.c.  of  water.  Acidulate  very  slightly  with  acetic  acid,  boil 
and  filter.  The  nitrate  should  be  water-clear.  Notice  the  brown 
color  of  the  coagulum.  To  what  is  it  due?  Evaporate  the  filtrate  to 
a  small  volume,  about  20  c.c.  If  a  precipitate  forms  during  the  evap- 
oration it  should  be  filtered  off.  Test  the  clear,  concentrated  liquid 
as  follows: 

(a).  Boil  some  Fehling's  solution  in  a  test-tube,  then  add  some 
of  the  liquid  and  boil  again.  A  yellowish-red  precipitate  of  cuprous 
oxide  indicates  the  presence  of  sugar. 

(b).  Acidulate  a  little  of  the  liquid  with  HN03  and  add  some 
AgN03.  A  heavy  white  precipitate  soluble  in  NH4OH  indicates  the 
presence  of  NaCl. 

(c).  Acidulate  another  portion  with  HN03  and  add  some  ammon- 
ium 'inolybdate  solution.  On  gentle  warming  a  yellowish  precipitate 
or  coloration  indicates  phosphates,  The  test  for  phosphoric  acid  can 
be  made  by  adding  NH4OH  to  the  liquid,  then  magnesia  mixture.  A 
white  cloud  or  precipitate  forms  if  phosphoric  acid  is  present. 

[d).  Evaporate  the  remainder  of  the  liquid  in  a  watch-glass  on 
a  water-bath  till  only  a  few  drops  remain.  Then  set  aside  to  cool  and 
examine  under  the  microscope  for  crystals  of  NaCl. 


BLOOD.  109 

Blood  Serum. 

Preparation. — The  blood  is  received  directly  from  an 
animal  into  a  wide  cylindrical  vessel  or  into  a  common 
fruit-  or  battery-jar.  It  clots  in  a  short  time,  forming-  a 
solid  coag'ulum.  The  vessel  is  then  placed  in  an  ice-chest 
for  36 — 48  hours.  As  the  clot  shrinks  the  clear  yellow 
serum  is  squeezed  out  and  collects  on  top.  This  yellow 
serum  is  removed  with  a  pipette  and  is  used  for  the  follow- 
ing" experiments.  It  not  infrequently  happens  that  the 
serum  as  obtained  is  reddish,  due  to  the  presence  of  blood 
corpuscles.  In  that  case  it  is  best  to  place  the  serum  in  a 
tall,  narrow  beaker  and  set  it  aside  in  the  ice-chest  for  1 — 2 
days  when  the  corpuscles  will  subside  and  leave  a  straw- 
yellow,  clear  serum  above. 

Blood  plasma,  the  liquid  portion  of  the  living  blood, 
contains  at  least  three  proteids — fibrinogen,  serum  albumin, 
and  serum  globulin.  In  the  process  of  clotting  the  fibrin- 
ogen is  changed  to  fibrin  and  hence  the  blood  serum  con- 
tains the  two  proteids  serum  albumin  and  serum  globulin, 
or  para-globulin. 

Carefully  review  in  this  connection  the  work  done  on 
the  proteids  of  blood  serum  (p.  47).  What  is  precipitated 
if  blood  serum  is  saturated  with  MgS04?     With  (NH4)2S04? 

1.— Determine  the  coagulating  point  of  undiluted  blood  serum 
(5 — 10  c.c.)  according-  to  the  method  given  under  egg-albumin,  Exp.  21, 
(p.  45).  Note  the  temperature  at  which  the  contents  of  the  tube 
become  cloudy:  when  they  gelatinize  and  when  they  become  solid. 

2. — In  each  of  three  tubes  place  1  c.c.  of  blood  serum.  To  tube  1 
add  nothing.  To  tubes  2  and  3  add  5  and  10  c.c.  respectively  of  dis- 
tilled water.  Immerse  in  a  boiling  water-bath  for  10  minutes.  Note 
the  result.     No.  1  coagulates  solid,  whereas  Nos.  2  and  3  do  not. 

Sufficient  dilution  of  serum  with  water  renders  it  non- 
coagulable  by  heat.     If   tap-water  is  used,  owing  to  the 


110  PHYSIOLOGICAL  CHEMISTRY. 

presence   of  calcium   salts,  partial   coagulation  will   take 
place. 

3. — To  each  of  four  tubes  add  1  c.c.  of  blood  serum;  then  add  to 
each  10  c.c.  of  distilled  water.  To  tubes  1  and  2  add  one  and  five 
drops  respectively  of  1  per  cent,  acetic  acid;  to  tube  3  add  a  couple  of 
drops  of  CaCl2  solution;  to  tube  4  add  two  g.  of  NaCl.  Immerse  the 
tubes  in  a  boiling  water-bath  for  10  minutes.  Note  the  results  and 
explain  the  same. 

As  shown  above  in  Exp.  2  blood  serum  diluted  with  10  parts  of 
water  does  not  coagulate  on  heating".  In  Exp.  3  tube  1  does  coagulate, 
whereas  tube  2  does  not.  To  tube  2  now  add  1  c.c.  of  a  10  per  cent. 
NaCl  solution  and  boil;  it  coagulates  at  once.  Tube  3  contains  a 
fibrinous  coagulum,  whereas  tube  4  coagulates  solid. 

What  effect  would  the  addition  of  NaCl  to  serum  have  on  the 
coagulating  point? 

Compare  carefully  this  and  the  preceding  experiment  with  Exp. 
7  and  8  (p.  41).  As  shown  before  even  slight  excess  of  acetic  acid 
tends  to  prevent  precipitation  of  albumin  and  globulin,  whereas  NaCl 
favors  precipitation. 

4. — To  5  c.c.  of  blood  serum  in  a  test-tube  add  1  drop  of  formalin, 
mix  and  boil.     The  blood  serum  does  not  coagulate. 

5. — To  45  c.c.  of  water  in  a  small  beaker  add  5  c.c.  of  blood  serum, 
mix  and  filter.  Receive  the  filtrate  in  a  50  c.c.  graduate  and  place 
this  in  a  beaker  of  cold  water.  Pass  a  current  of  C02  through  the 
diluted  serum  for  about  15  minutes.  Then  cork  and  set  aside  in  cold 
water  for  some  hours.  Para-globulin  is  thrown  out  of  solution  as  a 
fine  cloud  and  eventually  settles  to  the  bottom  as  a  white  precipitate. 

6.— To  50  c.c.  of  water  add  1  c.c.  of  blood  serum  and  mix.  To 
this  dilute  blood  serum  apply  the  following  tests: 

(a).  To  about  10  c.c.  of  the  diluted  serum  add  1 — 2  drops  of  strong 
HN03.  The  cloudiness  that  forms  disappears  on  shaking.  Now  heat 
the  contents  of  the  tube  to  boiling.  A  yellowish  color  develops,  but 
no  coagulation  takes  place.     Divide  the  liquid  into  two  portions. 

1. — Cool  one  portion,  then  add  5 — 6  drops  of  HN03  and  boil. 
Coagulation  results. 

2. — Raise  the  other  portion  to  boiling,  then  add  5 — 6  drops  of 
HNO3  and  boil.     Coagulation  likewise  results. 


BLOOD  111 

(b).  To  5  c.c.  of  the  diluted  serum  (1 — 50)  add  an  equal  volume  of 
water.  This  gives  a  serum  diluted  1—100.  To  this  very  dilute  serum 
add  a  drop  of  strong  HN03  and  boil.  No  coagulation.  Divide  the 
liquid  into  two  portions. 

1.— Cool  one  portion,  then  add  5 — 6  drops  of  HN03  and  boil.  It 
coagulates. 

2. — Raise  the  other  portion  to  boiling,  then  add  5 — 6  drops  of 
HN03  and  boil.     The  solution  remains  clear. 

Compare  these  two  experiments  and  explain  the  difference  in 
results.  The  precaution  that  should  be  taken  when  testing  for  small 
amounts  of  albumin  is  emphasized  in  the  next  two  experiments. 

7. — To  5  c.c.  of  the  diluted  serum  (1 — 50)  add  5 — 6  drops  of  strong 
HNO3.     A  cloudiness  forms  and  on  boiling  coagulation  takes  place. 

(«).  Repeat  this  experiment  with  serum  diluted  as  above  under  b 
{1—100).     Coagulation  takes  place  as  in  the  case  of  the  (1 — 50)  serum. 

8. — Boil  5  c.c.  of  the  dilute  serum  (1 — 50)  and  while  boiling  hot 
add  5 — 6  drops  of  HNOH.  Coagulation  results.  Compare  the  volume 
of  the  precipitate  with  that  obtained  in  Exp.  6  a  and  7. 

(a).  Repeat  this  experiment  with  a  serum  diluted  as  above 
under  b  (1 — 100).  Only  a  slight  precipitate  forms.  Compare  the  vol- 
ume of  the  precipitate  with  that  obtained  in  Exp.  6  b  and  7  a  . 
Explain. 

It  is  evident  from  the  above  experiments  that  in  the 
heat  and  HN03  test  for  albumin,  in  the  urine  or  elsewhere, 
it  is  necessary  to  take  into  account  the  amount  of  HNO, 
added  and  whether  the  solution  is  cold  or  hot.  The  best 
result  is  obtained  therefore,  when  albumin  is  present  in 
minute  quantities,  by  adding"  to  the  cold  solution  an  excess 
of  HNO3  (5 — 6  drops)  to  a  permanent  cloudiness  and  then 
boiling".     Maximum  coagulation  will  then  take  place. 

HN03  and  heat  will  coagulate  albumin  where  heat 
alone  will  fail  to  do  so.  This  may  be  the  case  if  the  urine 
tested  has  an  alkaline  reaction.  An  additional  advantage 
in  the  use  of  HN03  is  that  it  will  dissolve  any  phosphates 
that  may  be  thrown  out  of  solution  on  heating  the  urine. 

9. — To  5  c.c.  of  the  diluted  serum  (1—50)  add  1  drop  of  1  per  cent 


112  PHYSIOLOGICAL  CHEMISTRY. 

acetic  acid  (1  c.c.  of  glacial  acid  diluted  to  100  c.c.  with  water).  A 
cloudiness  results.  Test  the  reaction  of  the  liquid,  then  boil.  A 
coagulum  forms  and  the  liquid  is  perfectly  clear. 

(a).  Repeat  this  experiment,  first  raising"  the  dilute  serum  to 
the  boiling  point  and  then  adding  one  drop  of  the  1  per  cent,  acetic 
acid.     What  is  the  result? 

10. — To  5  c.c.  of  the  diluted  serum  (1 — 50)  add  3—4  drops  of  the 
dilute  acetic  acid  used  above.  Test  the  reaction  of  the  liquid,  then 
boil.     No  coagulation  takes  place,  but  the  liquid  is  opalescent. 

(a).  Boil  5  c.c.  of  the  diluted  serum  (1 — 50),  and  while  boiling  hot 
add  about  10  drops  of  1  per  cent,  acetic  acid  and  boil  again.  The 
cloudiness  that  forms  at  first  redissolves. 

In  precipitating  proteids,  from  urine  or  other  solutions, 
by  means  of  acetic  acid  and  heat,  care  must  therefore  be 
taken  to  add  the  acetic  acid  to  the  cold  solution  to  neutral- 
ization and  after  that  to  heat  to  boiling-.  Even  a  slight 
acidity  due  to  acetic  acid  will  keep  albumin  in  solution. 

Compare  the  behavior  of  acetic  and  nitric  acids  with 
the  serum  proteids. 

11.— To  some  of  the  dilute  serum  (1—50)  add  1—2  drops  of  HgCl2. 
A  white  precipitate  forms.  Shake  up  thoroughly  and  divide  into  two 
portions. 

(a).  To  one  portion  add  an  equal  volume  of  NaCl  solution  (1 — 10J. 
The  precipitate  promptly  dissolves. 

(6).  To  the  other  portion  add  an  equal  volume  of  undiluted 
serum.     The  precipitate  likewise  promptly  dissolves. 

The  precipitate  of  mercury  and  albumin  is  therefore 
soluble  in  NaCl,  also  in  excess  of  proteids.  Of  what  im- 
portance is  this  fact  in  practical  disinfection?  Compare 
this  test  with  the  similar  experiment  on  egg  albumin  (Exp. 
15,  p.  44).  Note  the  difference  in  the  behavior  of  the  two 
proteid  solutions. 

12.— To  some  of  the  dilute  serum  (1—50)  add  dilute  CuS04  solution 
till  a  precipitate  forms.  Then  add  a  few  drops  of  strong  NaOH  solu- 
tion (1 — 3).     The  precipitate  redissolves,  yielding  a  blue  solution. 


BLOOD.  113 

What  other  substances  give  similar  solutions  of  cupric 
hydrate? 

If  silver  nitrate  or  lead  acetate  be  added  to  the  dilute 
serum  what  would  be  the  result?  What  is  the  behavior  of 
the  salts  of  heavy  metals  with  proteids? 

The  reactions  given  by  serum  albumin  and  serum  glob- 
ulin as  worked  out  in  the  table  (p.  46)  will  of  course  be 
given  by  the  dilute  blood  serum. 

Fibrin. 

The  coagulum  obtained  by  whipping  freshly  drawn 
blood  is  cut  up  into  small  pieces  and  washed  in  running 
water  till  perfectly  white. 

Fibrin  on  contact  with  dilute  HC1  at  40°  swells  up  and 
the  contents  of  the  tube  become  solid  in  a  few  minutes 
(Exp.  1,  p.  71).  Solution  then  gradually  takes  place  so 
that  in  2 — 3  days  the  fibrin  disappears.  An  acid  albumin 
results  (Exp.  4,  p.  73). 

Fibrin  swells  up  also  in  5  percent,  oxalic  acid  solution, 
but  does  not  dissolve  readily.  It  is  also  soluble  in  dilute 
neutral  salt  solutions. 

Place  in  each  of  two  test-tubes  about  5  c.c.  of  hydrogen  perox- 
ide. To  one  add  a  shred  of  fresh  fibrin.  Oxygen  is  set  free  especially 
on  slight  warming,  through  so-called  catalytic  action.  This  action  is 
probably  due  to  remnants  of  leucocytes  (nucleo-histon). 

To  the  other  tube  add  some  boiled  fibrin.     What  is  the  result? 

*  Fibrinogen. 

In  a  two  liter  flask,  or  cylinder,  place  100  c.c.  of  a  6  per 
cent,  solution  of  potassium  oxalate.  Collect  into  the  flask 
about  two  liters  of  blood,  direct  from  the  animal,  and  mix 
thoroughly  at  once.  This  mixture  now  contains  about  0.3 
per  cent,  of  the  oxalate  which  precipitates  the  calcium  salts 
present  and  thus  prevents  coagulation. 


114  PHYSIOLOGICAL  CHEMISTRY. 

Centrifugate  the  blood  and  keep  the  clear  liquid  (oxal- 
ate plasma)  on  ice  for  24  hours  or  more.  The  deposit  that 
forms  is  largely  due  to  prothrombin,  the  parent-substance 
of  the  fibrin  ferment.  This  is  then  filtered  off.  To  one 
liter  of  clear  filtered  oxalate  plasma  add  one-half  its  vol- 
ume of  a  previously  filtered  saturated  sodium  chloride  solu- 
tion which  contains  y2 — 1  per  cent,  of  potassium  oxalate. 
Filter  off  and  discard  the  precipitate. 

To  the  new  filtrate  add  1- — \y2  liters  of  the  filtered 
saturated  NaCl  solution  containing-  potassium  oxalate. 
The  liquid  now  contains  for  one  volume  of  original  plasma 
two  volumes  of  the  salt  solution.  The  fibrinogen  separates 
and  floats  on  the  top  as  a  jelly-like  mass.  Remove  this 
mass  with  the  hand  and  squeeze  as  free  of  liquid  as  possi- 
ble. Then  place  it  in  a  6  to  8  per  cent,  solution  of  salt  con- 
taining oxalate  as  above.  Most  of  the  precipitate  dissolves. 
Decant  carefully  the  liquid,  keeping  back  the  foam  and 
undissolved  particles. 

This  solution  of  fibrinogen  is  now  purified  by  a  second 
precipitation  by  addition  of  an  equal  volume  of  the  satur- 
ated salt  solution  containing  oxalate.  The  fibrinogen  that 
is  thrown  out  of  solution  is  removed  as  before,  redissolved 
and  again  reprecipitated  as  above.  These  three  precipita- 
tions of  the  fibrinogen  will  yield  a  pure  product.  The  pre- 
cipitate is  finally  squeezed,  then  dissolved  in  distilled  water 
and  filtered.  The  solution  contains  pure  fibrinogen,  free 
from  calcium  (Hammarsten). 

*  Fibrin  Ferment. 

Blood  serum  is  saturated  with  magnesium  sulphate. 
The  filtrate  is  diluted  with  water  and  very  dilute  NaOH  is 
slowly  added,  while  constantly  stirring,  till  a  rather  abund- 
ant flocculent  precipitate  of  magnesium  hydrate  forms. 
This  precipitate  drags  down  mechanically  the  ferment. 
The  precipitate  is  collected,  washed,   squeezed  as  dry  as 


BLOOD.  115 

possible,  then  dissolved  in  water  by  addition  of  acetic  acid 
to  neutral  reaction.  The  magnesium  salts  are  removed  by 
dialysis  and  finally  the  last  traces  of  calcium  and  magne- 
sium are  removed  by  addition  of  potassium  oxalate  (j4 — 1 
per  cent.).  The  filtered  liquid  contains  the  fibrin  ferment, 
free  from  calcium,  and  when  added  to  the  fibrinogen  solu- 
tion prepared  as  above,  likewise  free  from  calcium,  a  pre- 
cipitate of  fibrin  promptly  forms. 


CHAPTER    IX. 


MILK. 


Milk  is  a  secretion  of  the  mammary  gland.  It  is  com- 
posed of  water,  casein,  globulin,  albumin,  fats,  milk-sugar, 
and  inorganic  salts.  The  color  of  milk  is  due  in  part  to 
the  suspended  fat  globules,  and  in  part  to  the  casein  which 
is  held  in  solution  by  calcium  phosphate.  The  specific  grav- 
ity of  milk,  from  a  single  animal,  may  vary  considerably; 
usually  from  1.028  to  1.035,  but  maybe  as  high  as  1.039. 
Market  milk  which  is  the  mixture  of  the  product  of  several 
animals  always  ranges  from  1.029  to  1.034.  The  average 
specific  gravity  of  whole  milk  is  placed  at  1.029. 

The  reaction  of  milk  is  usually  alkaline  or  amphoteric. 
It  may  however  be  acid,  and  this  is  especially  true  of  car- 
nivorous animals.  On  standing  milk  becomes  gradually 
acid  owing  to  the  formation  of  lactic  acid  by  fermentation. 
Fresh  milk  does  not  coagulate  on  heating.  After  fermen- 
tation sets  in  milk  will  coagulate  on  heating,  and  later 
curdles  without  the  application  of  heat.  Sterilized  milk, 
properly  kept,  will  remain  sweet  indefinitely.  The  scum 
which  forms  on  boiled  milk  is  not  coagulated  albumin  but 
a  combination  of  casein  and  calcium.  When  removed  a 
new  scum  forms  on  the  milk  when  heated  and  this  may  be 
repeated,  again  and  again.  Solutions  of  casein  under  simi- 
lar conditions  become  covered  with  scum. 

The  addition  of  rennet  to  milk  produces  in  a  short  time 
a  solid  coagulum,  the  curd  or  cheese.  The  clear  liquid 
remaining  is  the  whey,  or  milk-serum.     The  reaction  of  the 


MILK.  117 

milk  is  not  affected  by  this  change.  The  presence  of  cal- 
cium is  necessary  to  the  formation  of  curd.  The  casein 
originally  present  in  the  milk  is  apparently  changed  by  the 
ferment  into  two  proteids.  One  of  these  unites  with  cal- 
cium to  form  the  curd  and  is  known  as  para-casein.  The 
other  proteid  is  formed  in  small  amount,  is  related  to  the 
albumoses,  and  is  known  as  whey-proteid. 

Casein  is  a  complex  proteid  belonging  to  the  nucleo- 
albumins.  It  is  insoluble  in  water  but  is  readily  dissolved 
in  the  presence  of  alkalis.  A  solution  in  calcium  hydrate 
can  be  neutralized  with  phosphoric  acid  without  precipita- 
tion of  the  casein.  The  milky  liquid  thus  obtained  con- 
tains, in  solution  or  suspension,  the  casein  and  considera- 
ble calcium  phosphate.  Casein  is  thrown  out  of  solution 
by  dilute  acids,  or  by  saturation  with  NaCl  or  MgS04.  It 
is  also  precipitated  by  metallic  salts.  In  the  presence  of 
calcium  a  solution  of  casein  is  coagulated  by  rennet.  As  in 
the  case  of  milk,  a  solution  of  casein  when  boiled  becomes 
covered  with  a  scum.  On  digestion  with  pepsin  it  yields 
pseudo-nuclein  which  contains  phosphorus.  The  casein  in 
woman's  milk  is  different  from  that  in  cow's  milk.  The 
former  is  more  difficult  to  precipitate  with  acids,  salts  and 
rennet.  When  precipitated  by  an  acid  the  coagulum  is 
finely  fiocculent  and  dissolves  readily  in  an  excess  of  acid, 
whereas  casein  from  cow's  milk  is  coarsely  fiocculent  and 
is  less  readily  soluble  in  excess  of  acid.  Unlike  casein 
from  cow's  milk  it  does  not  yield  pseudo-nuclein  on  diges- 
tion. Casein  is  derived  apparently  from  a  nucleo-proteid 
contained  in  the  protoplasm  of  the  cells  of  the  gland. 

The  globulin  of  milk,  or  lacto-globulin  of  Sebelien,  is 
probably  identical  with  serum  globulin.  Lacto-albumin  is 
related  to,  but  not  identical  with,  serum  albumin.  Like 
casein  and  milk-sugar  it  is  a  special  product  of  the  cells  of 
the  gland.  Schlossmann  found  the  three  proteids  present 
in  milk  in  the  following  quantities:  Casein,  3.19  per  cent.; 
albumin,  0.37  per  cent. ;  globulin,  0.15  per  cent.    Only  traces 


118  PHYSIOLOGICAL  CHEMISTRY. 

of  urea,  creatin,  etc. ,  are  normally  present  in  milk,  conse- 
quently all  the  nitrogen  present  can  be  considered  as  con- 
tained in  the  proteid  substances. 

The  fat  is  present  as  an  emulsion  of  fat  globules. 
These  vary  in  size  in  milk  from  the  same  species,  and  from 
different  species.  According"  to  Woll  they  are  on  an  aver- 
age 3.7  p  in  diameter,  and  from  1  to  5.7  millions  of  these  glob- 
ules are  contained  in  1  c.c.  of  milk.  The  former  belief 
that  the  fatty  globules  were  surrounded  by  an  albuminous 
envelope  is  no  longer  held.  The  fat  is  supposed  to  result 
from  a  degeneration  of  the  protoplasm  of  the  cells,  but  it  is 
possible  that  a  part,  at  least,  is  brought  to  the  gland  by 
the  blood. 

The  sugar  present  in  the  milk,  lactose,  is  a  specific 
product  of  the  gland  cells  and  is  not  directly  derived  from 
the  blood.  It  is  possible  that  it  is  derived,  like  casein, 
from  the  nucleo-proteids  in  the  cells.  That  these  com- 
pounds can  give  rise  to  carbohydrates  has  been  demon- 
strated. In  exceptional  cases  milk-sugar  may  appear  in 
the  urine.  Like  glucose  it  is  dextro-rotatory  and  reduces 
Fehling's  solution.  Although  readily  decomposed  by  bac- 
teria it  is  not  acted  upon  by  pure  yeast.  This  fact  as  well 
as  its  solubility,  crystalline  form  and  the  formation  of 
mucic  acid  on  oxidation  with  nitric  acid  distinguishes  lac- 
tose from  glucose. 

The  colostrum  corpuscles  can  be  considered  as  epithe- 
lial cells 'which  have  taken  up  fatty  globules,  rather  than 
as  degenerated  cells.  They  are  found  in  milk  secreted  just 
before  and  after  delivery.  And  appear  as  nucleated,  gran- 
ular cells,  containing  numerous  fatty  granules.  They  are 
from  5  to  25  n  in  diameter.  The  milk  at  this  time  is  yellow- 
ish in  color,  alkaline  in  reaction,  and  has  a  high  specific 
gravity  1.046 — 1.080.  When  such  milk  is  heated  it  coagu- 
lates solid  owing  to  the  presence  of  increased  quantities  of 
albumin  and  globulin.  (See  table  giving  composition  of 
colostrum,  Chapter  XI). 


MILK.  11 

1. — Examine  a  drop  of  milk  under  the  microscope.  Sketch  the 
different  sized  globules  present  and  measure  their  diameter.  They 
average  about  5  /',  but  some  globules  may  attain  a  diameter  of  18  (i  or 
more. 

2.— Examine  microscopically  a  drop  of  skimmed  milk.  What 
difference  is  observed  between  this  and  whole  milk? 

3. — Examine  with  a  microscope  colostrum  milk.  Sketch  and 
measure  the  colostrum  corpuscles. 

4. — Place  about  10  c.c.  of  milk  in  a  test-tube  and  boil.  Then 
immerse  litmus  paper  in  the  hot  milk  for  1 — 2  minutes,  remove  and 
examine.     Under  what  conditions  does  milk  become  acid? 

5. — Boil  about  25  c.c.  of  milk  in  a  small  beaker  for  five  minutes. 
No  coagulation  proper,  but  a  scum  may  form.  Remove  the  scum  with 
a  spoon  or  spatula  and  heat  again;  a  new  scum  forms.  This  formation 
of  scum  will  repeatedly  take  place.  What  is  the  nature  of  this  scum? 
Casein  is  not  coagulated  by  heat.  Why  does  not  the  albumin  in  the 
milk  coagulate?     Save  the  milk  for  Exp.  13. 

6.— To  about  10  c.c.  of  milk  in  a  test-tube  add  one  drop  of  dilute 
acetic  acid  (1—10),  then  boil.  The  casein  is  coagulated  and  carries 
down  with  it  the  fat.     The  serum  is  clear. 

7. — Set  aside  in  a  test-tube  some  milk  over  night  at  ordinary 
room  temperature.  The  next  day  heat  the  contents  to  boiling.  Ex- 
plain the  result. 

8. — Place  10  c.c.  of  milk  in  each  of  five  test-tubes. 

To  No.  1  add  y2  c.c.  of  very  dilute  HC1  (10  drops  of  HC1  to  50  c.c 
of  water). 

To  No.  2  add  yz  c.c.  of  2  per  cent.  Na20O3  solution. 

To  No.  3  add  %  c.c.  of  saturated  (NHJ2C204  solution  (1—20). 

Then  add  to  each  of  these  three  tubes  and  also  to  Nos.  4  and  5 
two  drops  of  rennet  solution  and  mix.  Heat  the  contents  of  tube 
No.  5  to  boiling.  Then  place  all  the  tubes  in  a  water-bath  at  40°  and 
examine  every  3 — 5  minutes. 

The  contents  of  tube  1  will  coagulate  in  a  few  minutes;  No.  4  next; 
Nos.  2,  3,  and  5  will  not  coagulate.  The  latter  does  not  because  the 
heat  has  destroyed  the  ferment.  The  action  of  rennet  is  retarded  or 
prevented  by  alkali,  and  is  favored  by  acid,  such  as  is  present  in  the 
gastric  juice. 

9 


120  PHYSIOLOGICAL  CHEMISTRY. 

The  coagulum  which  forms  contains  para-casein  and 
the  fat.  The  clear  liquid  that  separates  from  the  coagu- 
lum  on  standing  is  the  whey,  or  milk-serum.  Para-casein  is 
different  chemically  from  the  casein  obtained  by  the  addi- 
tion of  an  acid  to  milk.  Calcium  salts  must  be  present  in 
order  that  para-casein  may  form.  Tube  No.  3  does  not 
coagulate  because  the  calcium  is  thrown  out  of  solution  as 
the  oxalate.  Compare  the  change  that  takes  place  with 
that  in  the  clotting  of  blood.  If  oxalate  of  sodium  is  added 
to  freshly  drawn  blood  what  is  the  result? 

8  a. — Continue  heating  tube  3  at  40°  for  about  )4  hour.  Then  add 
2 — 3  drops  of  CaCl2  solution.  The  liquid  instantly  solidifies.  This  shows 
that  the  rennet  has  acted  on  the  casein  and  changed  it  into  the  modi- 
fication which,  with  calcium,  yields  para-casein. 

Calcium  is  likewise  necessary  to  the  coagulation  of 
blood,  not  however,  for  the  formation  of  clot  directly  as  in 
the  case  of  the  milk  curd.  Calcium-free  blood  plasma  (oxal- 
ate plasma)  and  calcium-free  fibrin  ferment  when  mixed, 
promptly  yield  a  clot  of  fibrin.  The  calcium  is  necessary 
to  the  formation  of  the  fibrin  ferment  from  a  parent  sub- 
stance, prothrombin  (Hammarsten). 

9. — To  some  milk  in  a  test-tube  add  1 — 2  volumes  of  ether,  close 
and  shake  thoroughly.  The  fat  globules  do  not  dissolve;  the  milk 
remains  opaque.  Now  add  a  few  drops  of  NaOH  and  shake  again. 
The  ether  now  dissolves  the  fat  and  the  liquid  clears  up.  This  reac- 
tion was  taken  at  one  time  to  indicate  that  the  globules  were  sur- 
rounded by  an  albuminous  envelope.  Compare  this  test  with  the 
action  of  ether  on  blood. 

10. — To  some  milk  in  a  test-tube  add  a  few  drops  of  NaOH  and 
heat.     The  liquid  becomes  yellow,  then  orange,  and  finally  brown. 

11. — To  a  4  per  cent,  solution  of  lactose  add  a  little  NaOH  and 
heat.  The  same  color  reaction  is  developed  as  in  Exp.  10,  which  is  due. 
to  the  sugar  present  in  the  milk. 

12. — To  some  milk  add  tincture  of  guajac  and  mix:  then  pour  on 
a  layer  of  old  turpentine.  A  deep  blue  color  develops.  This  test  is 
also  given  by  blood. 


MILK.  121 

13. — Repeat  the  preceding  experiment  using,  however,  the  boiled 
milk  from  Exp.  5.  The  color  does  not  develop.  Heat  has  changed  the 
proteids  so  that  they  can  no  longer  assist  in  the  oxidation  of  the 
guajac  resin. 

14. — To  about  10  c.c.  of  milk  in  a  test-tube  add  5  g.  of  powdered 
MgS04  and  shake  thoroughly.  Then  pour  onto  a  filter,  resting  in  a 
test-tube,  and  set  aside  to  filter  over  night.  Boil  the  clear  filtrate — 
albumin  coagulates.  The  casein  is  precipitated  almost  completely 
by  MgS04. 

15.— To  5  c.c.  of  milk  add  4  volumes  (20  c.c.)  of  strong  alcohol, 
shake  thoroughly  and  set  aside.  All  the  proteids  present  are  pre- 
cipitated. 

16. — Dilute  10  c.c.  of  milk  with  about  30  c.c.  of  water  and  divide 
into  three  portions. 

To  1  add  1—2  c.c.  of  potassium  alum  solution  (1 — 10)  and  shake. 
The  casein  is  precipitated  and  carries  down  with  it  the  fat. 

To  2  add  1 — 2  c.c.  of  copper  sulphate  solution  (1 — 10)  and  shake. 
A  voluminous  greenish  blue  precipitate  of  the  proteids  present 
(and  fat)  results. 

To  3  add  about  2  c.c.  of  Almen's  tannic  acid  solution  and  shake. 
The  proteids  are  precipitated. 

*17. — Moisten  a  few  granules  of  pepsin  with  a  drop  of 
water,  or  better  with  a  drop  of  a  0.7  per  cent.  NaCl  solu- 
tion. Then  add  5  c.c.  of  milk,  mix  and  set  aside  in  a  water- 
bath  at  40°.  Coagulation  results  in  a  few  minutes.  It  will 
fail  if  more  water  or  salt  solution  is  added  to  the  pepsin 
(Pekelharing).  Chymosin  on  digestion  with  pepsin  and 
0.3  per  cent.  HC1  is  destroyed  (Hammarsten). 

18. — Add  50  c.c.  of  milk  to  about  400  c.c.  of  water,  mix  well  and 
while  stirring  add  dilute  acetic  acid  (1 — 10),  drop  by  drop,  till  the  pre- 
cipitate becomes  coarsely  flocculent  and  ceases  to  increase.  Stir 
thoroughly  and  set  aside  over  night.  The  reaction  should  be  dis- 
tinctly acid. 

The  precipitate  consists  of  casein  and  fat.  Filter  off  the  precip- 
itate and  allow  to  drain  well,  then  fold  over  half  the  filter  in  the 
funnel  and  apply  gentle  pressure  with  the  fingers  until  no  more  water 
can  be  squeezed  out. 


122  PHYSIOLOGICAL  CHEMISTRY. 

Transfer  the  precipitate  to  a  small  dry  beaker,  add  about  30  c.c. 
of  strong"  alcohol  and  stir  thoroughly  so  as  to  dehydrate  the  casein. 
Then  filter  and  again  squeeze  the  contents  of  the  filter  as  dry  as  pos- 
sible. Transfer  the  precipitate  to  a  small  dry  beaker,  add  about 
50  c.c.  of  ether  and  heat  on  a  warm  water-bath  with  constant  stirring 
for  about  10  minutes.  Owing  to  danger,  the  light  should  be  very  low 
or  better  turned  out.  Finally  transfer  the  contents  to  a  filter  and 
squeeze  as  dry  as  possible. 

Spread  open  the  filter  on  the  table,  allow  the  remaining  ether  to 
evaporate,  then  powder.     The  white  chalky  powder  is  casein. 

The  ether  filtrate  received  in  a  small  beaker  or  evaporating  dish 
and  evaporated  cautiously  on  the  water-bath  gives  the  milk-fat. 

The  aqueous  filtrate  from  the  casein  and  fat  precipitate  con- 
tains albumin  and  milk-sugar.  Place  it  in  a  beaker  and  boil  for  15 
minutes.  Filter  off  the  precipitate  of  albumin  and  reserve  for  subse- 
quent tests. 

Concentrate  the  filtrate  from  the  albumin,  in  a  beaker  on  a  wire 
gauze,  till  it  becomes  cloudy  and  bumps.  Cool  the  liquid;  the  cloudi- 
ness disappears  and  is  therefore  due  to  phosphates.  Heat  again  to 
boiling  and  filter  hot.  Concentrate  the  filtrate  now  on  the  water- 
bath  to  a  syrupy  consistency  and  set  aside  over  night.  Crystals  of 
milk-sugar  separate  on  standing. 

To  the  casein  obtained  in  the  above,  apply  the  biuret,  the  MiL 
Ion,  and  the  xanthoproteic  reactions.  Also  dissolve  a  portion  in  water 
to  which  some  Na2C03  solution  has  been  added.  Observe  the  cloudiness 
of  the  solution.  Heat  a  portion  of  the  casein  with  alkaline  lead 
acetate.  What  is  the  result?  Apply  the  same  tests  to  the  spe  cimen 
of  albumin.     Review  carefully  the  reactions  for  lactose  and  fats. 


CHAPTER     X. 

URINE. 

To  some  normal  urine  apply  the  following-  reactions 
and  carefully  note  the  results: 

1. — Test  the  reaction  with  litmus-paper.     What  is  it? 

2. — Heat  some  urine  with  a  strong-  acid  as  HC1,  HN03,  or  H2S04. 
Observe  the  peculiar  odor  that  is  emitted  and  the  change  in  color. 

3. — Concentrate  some  urine  in  a  small  dish  or  porcelain  crucible 
on  the  water-bath,  ignite  and  test  the  residue  by  means  of  a  platinum 
wire  and  flame  for  potassium  and  sodium. 

4. — Add  a  few  drops  of  oxalic  acid  or  ammonium  oxalate  solution 
to  some  urine.  Examine  the  precipitate  under  the  microscope  and 
test  its  solubility  with  acetic  and  hydrochloric  acids.     What  is  it? 

5. — To  some  urine  acidified  with  nitric  acid,  add  a  few  drops  of 
silver  nitrate  solution.  What  is  the  nature  of  the  precipitate?  Test 
its  solubility  with  ammonium  hydrate  and  with  nitric  acid.  Notice 
the  size  of  the  precipitate. 

6. — To  some  urine  acidified  with  hydrochloric  acid,  add  barium 
chloride.  What  does  the  precipitate  indicate?  Test  its  solubility  in 
acids.     How  large  is  the  deposit? 

7. — To  some  urine  add  uranium  acetate  solution.  The  yellowish 
white  precipitate  is  uranium  phosphate.  Test  its  solubility  in  min- 
eral acids,  and  in  acetic  acid. 

8. — Add  a  few  drops  of  ferric  chloride  to  some  urine.  What  does 
the  precipitate  consist  of? 

9. — Heat  some  of  the  urine  in  a  test-tube.  If  strongly  acid  no 
change  results,  but  if  it  is  feebly  acid  or  neutral  the  phosphate  of 
calcium  is  precipitated.  Why?  Write  the  formula  of  this  salt.  To 
a  portion  of  the  urine  with  this  precipitate  add  nitric  acid;  cool 
another  portion.     What  is  the  result?    - 


124  PHYSIOLOGICAL  CHEMISTRY. 

10. — To  some  urine  add  ammonium  hydrate — ammonium  magne- 
sium phosphate  and  calcium  phosphate  are  thrown  down.  When  the 
precipitate  subsides  examine  it  under  the  microscope.  What  is  the 
form  of  the  calcium  phosphate?  What  is  the  form  of  the  crystals  of 
the  ammonium  magnesium  phosphate?  To  a  portion  of  the  precipi- 
tate add  acetic  acid,  to  another  portion  hydrochloric  acid.  Note  the 
result. 

Filter  a  third  portion  and  to  the  filtrate  add  a  solution  of  mag- 
nesium sulphate.  A  precipitate  of  magnesium  ammonium  phosphate 
forms.  How  does  it  compare  in  bulk  with  that  obtained  above.  Ex- 
plain the  formation  of  this  precipitate.  What  is  meant  by  earthy 
phosphates;  by  alkali  phosphates? 

11. — Render  some  urine  alkaline  with  sodium  or  potassium 
hydrate.  The  phosphates  of  calcium  and  magnesium  are  precipi- 
tated. Examine  the  precipitate  under  the  microscope;  then  test  its 
solubility  in  acetic  and  hydrochloric  acids.     What  are  the  results? 

12. — Add  a  few  drops  of  mercuric  nitrate  solution  to  some  urine. 
Observe  that  the  precipitate  formed  by  the  first  drop  redissolves  on 
shaking;  that  when  an  excess  is  added  the  precipitate  is  permanent. 
To  a  portion  of  the  precipitate  add  some  sodium  chloride  solution;  to 
another  portion  add  nitric  acid.  Note  the  result.  The  precipitate 
contains  urea. 

13.— To  about  100  c.c.  of  urine  in  a  beaker  add  about  10  c.c. 
of  hydrochloric  acid  and  then  set  aside  for  24  hours.  The  slight  pre- 
cipitate of  reddish  crystals  consists  of  uric  acid.  Examine  under  the 
microscope. 

14. — Boil  some  urine  with  Fehling's  solution.    What  is  the  result? 

15. — Add  a  few  drops  of  picric  acid  solution  to  some  urine.  Note 
the  presence,  or  absence  of  a  precipitate.     What  is  it? 

16. — To  some  urine  add  acetic  acid,  then  a  few  drops  of  potas- 
sium f errocyanide.     Examine  the  same  as  under  15. 

17. — Heat  some  urine  with  Millon's  reagent.  A  red  color  would 
indicate  the  presence  of  what  substance? 

18. — To  5  c.c.  of  sulphuric  acid  add  about  10  c.c.  of  urine — a  gar- 
net red  color  is  ascribed  to  "urophaein,"  a  humin  substance. 


URINE.  125 

NH, 
Urea,  CH4N20,   =  CO     . 
NH2 

Urea  can  be  considered  as  an  amide  of  carbonic  acid 
CO(OH),,  and  is  therefore  spoken  of  as  carbamide.  It  can 
be  prepared  artificially  by  the  action  of  ammonia  on  car- 
bonyl  chloride  (CO.CL);  by  heating  solutions  of  ammonium 
cyanate;  by  hydration  of  proteins,  creatin,  uric  acid,  etc. 

It  is  the  chief  form  in  which  waste  nitrogen  leaves  the 
body.  The  nitrogen  present  in  the  complex  proteins,  de- 
rived from  the  food  and  present  in  the  fluids  and  cells  of  the 
body,  when  disintegration  results  passes  through  a  series  of 
successive  cleavage  products,  each  one  more  simple  than 
the  preceding,  and  eventually  appears  in  the  urine  as  urea 
or  as  other  waste  nitrogenous  substances.  From  82 — 88 
per  cent,  of  the  total  nitrogen  excreted  is  eliminated  as 
urea.  The  remaining  12 — 18  per  cent,  of  nitrogen  is  con- 
tained in  ammonia,  creatinin,  uric  acid,  xanthin  bases, 
indol  and  a  variety  of  other  compounds.  It  follows  there- 
fore that  the  total  amount  of  urea  in  a  day's  urine  can  be 
taken  as  a  measure  of  tissue  metabolism,  and  consequently 
is  of  great  clinical  significance. 

While  the  above  is  true  with  reference  to  man  and 
mammals  in  general,  it  does  not  hold  true  for  birds,  rep- 
tiles and  amphibians,  where  uric  acid  may  be  considered  as 
the  chief  nitrogenous  waste  product.  Urea  is  found  in  min- 
ute amount  in  the  blood,  lymph,  spleen,  and  liver,  but  not 
in  muscles.  When  on  account  of  a  disease  of  the  kidneys 
it  cannot  be  eliminated  from  the  body,  it  is  greatly  increased 
in  the  blood  and  elsewhere  in  the  body  and  may  then  ap- 
pear in  the  sweat,  vomit,  and  in  intestinal  contents. 

The  original  source  of  urea  is  the  protein  matter  of  the 
foods  and  tissues.  In  an  individual  possessing  a  constant 
weight,  as  an  adult,  the  total  nitrogen  in  the  food,  or  an 
amount  corresponding  to  that,  is  eliminated  by  the  kidneys 


126  PHYSIOLOGICAL  CHEMISTRY. 

within  24  hours  as  waste  nitrogen.  Some  of  this  waste 
nitrogen  naturally  results  from  the  destruction  and  disinte- 
gration of  the  tissues  of  the  body,  and  of  haemoglobin.  The 
remainder  probably  results  from  the  direct  breaking  down 
of  circulating  proteins.  It  has  been  supposed  that  this  dis- 
integration of  proteins  into  urea  could  not  be  accomplished 
except  through  the  aid  of  the  living  cell.  The  studies  of 
Drechsel  have  shown,  however,  that  it  is  possible  to  obtain 
urea  by  hydration  from  proteins,  whether  derived  from  ani- 
mals, or  from  plants. 

The  immediate  antecedents  of  urea  have  furnished  a 
fruitful  subject  for  investigation.  Apart  from  the  possi- 
bility of  making  urea,  by  hydration,  directly  from  proteids 
and  from  creatin,  it  may  be  said  that  urea  results  from  one 
of  the  following  antecedents: 

Amido  acids. 
Ammonium  carbonate. 
Ammonium  carbamate. 

It  is  well  known  that  amido  acids,  as  leucin,  glycocoll, 
etc.,  when  fed  to  animals,  increase  the  urea  in  the  urine. 
Furthermore,  in  certain  diseases  of  the  liver,  amido  acids 
are  increased,  whereas  urea  is  decreased.  When  blood 
containing  amido-acids  is  passed  through  a  freshly  re- 
moved liver  urea  is  increased.  It  is  certain  therefore 
that  these  acids  may  be  antecedents  of  urea.  They  are 
probably,  however,  not  changed  directly  into  urea,  but 
first  into  ammonium  carbonate.  In  general,  salts  of  or- 
ganic acids  are  oxidized  in  the  body  to  the  correspond- 
ing carbonates.  Thus  potassium  acetate,  if  administered, 
appears  in  the  urine  as  potassium  carbonate.  Ammonium 
acetate  does  not  appear  in  the  urine  as  the  carbonate  but 
as  urea.  Hence  amido  acids,  as  amido-acetic  acid,  are 
changed  first  into  ammonium  carbonate,  then  into  urea. 

As  stated  ammonium  carbonate  is  likewise  an  antece- 


URINE.  127 

dent  of  urea.  When  this  salt  is  administered  urea  is  in- 
creased in  the  urine.  The  same  is  true  of  the  ammonium 
salts  of  organic  acids  since  these  are  oxidized  in  the  body- 
to  the  carbonate,  and  the  result  is  therefore  the  same  as  if 
this  compound  was  taken  directly.  Furthermore,  if  am- 
monium carbonate  is  passed  through  a  perfectly  fresh  ex- 
cised liver  urea  will  be  formed. 

The  formation  of  urea  from  amido  acids,  and  from  am- 
monium carbonate  can  further  be  explained  by  the  forma- 
tion of  ammonium  carbamate  which  may  be  considered  as 
the  immediate  antecedent  of  urea.  The  relation  of  these 
three  bodies  can  be  seen  from  the  following-  formulae: 

/ONH4  /NH2  /NH2 

CO  CO  CO 

■\ONH4  \ONH4  \NH2 

Ammonium  Ammonium  Urea, 

carbonate.  carbamate. 

It  would  appear  that  the  removal  of  the  elements  of 
one  molecule  of  water  from  ammonium  carbonate  yields 
the  carbamate,  and  the  removal  of  a  second  molecule 
of  water  yields  urea.  Ammonium  carbamate  is  present 
in  the  urine  of  certain  animals,  and  is  notably  in- 
creased after  administration  of  lime  water.  The  lime, 
it  seems,  unites  with  carbamic  acid  in  the  body  and 
protects  this  against  conversion  into  urea.  When  the 
blood  of  the  portal  vein,  instead  of  passing  through  the 
liver,  is  directed  into  the  vena  cava,  the  liver  to  all  inten- 
tional purposes  is  removed  from  the  body.  Consequently 
the  antecedents  of  urea  are  not  all  changed  into  urea  and 
hence  appear  in  the  urine.  Carbamic  acid  appears  under 
these  conditions  in  the  urine  and  the  symptoms  of  intoxi- 
cation which  eventually  develop  are  probably  due  to  car- 
bamates. 

The  liver  is  probably  the  chief,  if  not  the  only  organ  in 
man  where  urea  is  made  out  of  its  antecedents.  This  is 
seen  from  the  fact  that  amido   acids   or   ammonium  car- 


128  PHYSIOLOGICAL  CHEMISTRY. 

bonate  when  passed  through  an  excised  liver  yield  urea. 
Furthermore,  in  structural  diseases  of  the  liver,  as  in  acute 
yellow  atrophy,  urea  is  diminished  considerably  and  is  re- 
placed by  its  antecedents,  ammonia,  amido  acids,  carbamic 
acid. 

The  urea  which  is  made  in  the  liver  is  carried  by  the 
blood  to  the  kidneys  and  there  excreted.  The  epithelial 
cells  lining-  the  convoluted  tubules  are  engaged  in  the 
active  excretion  of  urea.  If,  as  the  result  of  inflammatory 
changes,  these  cells  are  altered  or  destroyed,  urea  cannot  be 
eliminated  from  the  blood  and  hence,  with  other  waste  pro- 
ducts, accumulates  in  the  blood  and  elsewhere  and  leads  to 
intoxication — urcemia. 

The  amount  of  urea  excreted  in  24  hours  by  a  healthy 
adult  varies  on  an  average  from  25  to  30  grammes.  Women 
excrete  somewhat  less;  children,  in  proportion  to  their  body- 
weight,  excrete  relatively  more  than  adults.  In  old  age 
the  excretion  of  urea  is  diminished. 

The  most  important  factor  influencing  the  amount  of 
urea  excreted  is  the  kind  and  quantity  of  food  taken. 
Thus,  with  a  meat  diet  a  person  may  excrete  67  g.  of  urea, 
whereas  with  a  bread  diet  the  amount  may  drop  to  20  g.  It 
is  for  this  reason  that  urea  is  most  abundant  in  the  urine  of 
carnivorous  animals,  and  is  less  abundant  in  that  of  herbiv- 
orous animals.  In  starvation,  as  long  as  the  fats  and  car- 
bohydrates stored  up  in  the  body  last  the  amount  of  urea  is 
kept  low,  as  in  a  mixed  diet.  When  these,  however,  have 
all  been  consumed  the  animal  must  live  on  the  proteins  of 
its  tissues  exclusively  and  urea  is  at  once  increased  just  as 
if  an  exclusively  meat  diet  was  given.  Under  these  condi- 
tions the  animal  naturally  soon  perishes. 

Urea  is  not  increased  by  any  ordinary  amount  of  mus- 
cular exercise.  In  excessive  exercise,  pushed  to  the  verge 
of  exhaustion,  urea  is  increased  for  much  the  same  reasons 
as  in  starvation. 


URINE.  129 

Under  pathological  conditions  urea  may  be  increased 
considerably.  This  is  especially  seen  in  fevers  where, 
although  the  amount  of  food  taken  is  very  small,  the 
amount  of  urea  excreted  may  reach  50  g.  per  day  or  even 
more.  The  tissue  proteins  are  being  actively  broken  down 
and  hence  urea  is  formed.  The  fever  cannot  be  considered 
as  the  cause  of  this  rapid  disintegration  but  is  rather 
itself  a  result.  Thus,  whenever  complex  substances  are 
broken  down,  as  in  the  fermentation  of  sugar,  heat  is 
always  liberated.  In  diabetes  the  urea  may  be  increased; 
even  100  g.  may  be  daily  excreted. 

A  pathological  decrease  of  urea  is  of  much  greater 
importance.  A  decrease  is  met  with  in  but  one  febrile  dis- 
ease, namely  acute  yellow  atrophy  of  the  liver.  In  this 
organ  urea  is  made  and  hence  when  diseased  the  amount  of 
urea  in  the  urine  at  once  falls.  For  this  reason  all  struc- 
tural diseases  of  the  liver  are  accompanied  by  a  decrease 
in  urea.  Since  the  kidney  is  the  organ  whereby  urea  is 
eliminated  it  follows  that  in  structural  diseases  of  the  kid- 
neys this  elimination  will  be  decreased  or  even  sup- 
pressed. In  that  case  urea  accumulates  in  the  blood  and 
tissues  and  is  partially  excreted  by  the  sweat,  vomit  and 
intestinal  discharges.  Eventually  marked  intoxication 
(uraemia)  and  death  result.  Poisoning  will  follow  either 
from  non- elimination  of  urea  and  other  waste  products;  or 
from  non-formation  of  urea. 

1.  —  Preparation  from  the  urine. — Concentrate  about  500  c.c.  of 
urine  on  a  water-bath  to  a  thin  syrup;  cool  this  by  immersion  in  ice- 
water  and  then  add,  at  the  same  time  stirring-  well,  about  three  times 
its  volume  of  strong  nitric  acid  (1.3  specific  gravity)  which  previously 
has  been  boiled  to  expel  nitrous  acid  and  then  cooled  to  0°.  Allow 
the  mixture  to  stand  several  hours  at  a  low  temperature — 0°  is  best. 
Transfer  the  crystalline  mass  of  urea  nitrate  which  separates  out  to 
an  asbestos  filter  (glass-wool  or  sand),  wash  several  times  with  small 
amounts  of  ice-cold,  pure  concentrated  nitric  acid,  then  dissolve  in 
the  smallest  possible  amount  of  hot  water;  cool  again  and  precipitate 
with  concentrated  nitric  acid.     Drain  the  crystals  on  a  filter  as  above; 


130  PHYSIOLOGICAL  CHEMISTRY. 

dissolve  in  hot  water  arid  treat  with  a  small  quantity  of  pure  freshly 
precipitated  barium  carbonate  until  effervesence  ceases  and  the  solu- 
tion reacts  neutral.  Evaporate  on  a  water-bath  to  dryness,  pulverize 
the  residue  and  extract  it  repeatedly  with  cold  absolute  alcohol 
whereby  the  urea  is  dissolved  and  the  barium  salts  are  left  behind* 
Filter,  and,  if  necessary,  decolor  the  alcoholic  filtrate  by  boiling-  with 
animal  charcoal,  then  concentrate  to  a  small  volume  and  set  aside 
for  crystals  to  form. 

Write  out  equations  for 

1.  Urea  +  nitric  acid  = 

2.  Urea  nitrate  -\-  barium  carbonate  = 

2. — Synthetic  preparation. — Rub  up  in  a  mortar  10  g.  of  thoroughly 
dehydrated  potassium  ferrocyanide  with  3.75  g.  of  anhydrous  potas- 
sium carbonate,  transfer  to  an  iron  crucible,  cover  and  heat  over 
a  Bunsen  burner  or  blast-lamp  till  perfect  fusion  results.  To 
the  somewhat  cooled  but  still  fluid  mass  add  slowly  and  in  small 
quantities  18.74  g.  of  well  dried  red  lead,  then  heat  for  about  ten 
minutes,  at  times  stirring  thoroughly  with  a  glass  rod,  and  finally 
pour  the  mass  out  on  an  iron  plate.  Pulverize  the  cooled  mass  and 
dissolve  in  about  21  c.c.  of  water;  filter  the  solution  of  potassium 
cyanate  thus  obtained,  directly  into  an  evaporating  dish  containing  a 
solution  of  10  g.  of  ammonium  sulphate  in  about  15  c.c.  water.  The 
potassium  cyanate  is  converted  into  ammonium  cyanate.  Evaporate 
the  combined  aqueous  solutions  on  a  water-bath  to  dryness.  As  a 
result  of  the  heating  the  ammonium  cyanate  undergoes  molecular 
transposition  and  becomes  converted  into  its  isomer — urea  or  carba- 
mide. Extract  the  residue  several  times  with  warm  absolute  alcohol, 
filter  and  evaporate  the  combined  alcoholic  filtrates  on  a  water-bath 
almost  to  dryness,  then  set  aside  to  crystallize.  Purify,  if  necessary, 
by  re-crystallizing  several  times  from  alcohol. 

Write  out  the  equations  representing  the  three  stages  in  the 
above  process. 

1.  4  K4Fe(CN)6  +  4  K2C03  +  5  Pb304  = 

2.  2  CNOK  +  (NH4)2S04  = 

3.  CN.ONH,  = 

Why  do  we  add  potassium  carbonate?    Why  red  lead? 

Write  equations  showing  the  change  that  takes  place  when 
potassium  ferrocyanide  is  heated  by  itself;  when  it  is  heated  with 
potassium  carbonate. 


URINE.  131 

^Synthetic  preparation  (Volhard,  Ann.  259,  377). — Dis- 
solve 3.9  g.  of  potassium  cyanide  and  1  g.  of  potassium 
hydrate  in  100  c.c.  of  water,  and  add  slowly  a  solution  of 
6.3  g.  of  potassium  permanganate  in  100  c.c.  of  water;  keep 
the  temperature  below  17°.  Now  add  10  g.  of  ammonium 
sulphate  and  warm;  then  filter  off  the  manganese  dioxide, 
evaporate  the  filtrate  to  dryness  and  extract  the  residue 
with  95  per  cent,  alcohol;  concentrate  the  alcoholic  solution 
to  crystallization.  To  remove  traces  of  ammonium  chloride 
dissolve  the  urea  in  a  little  water,  add  some  barium  car- 
bonate, evaporate  and  extract  the  residue  with  absolute 
alcohol. 

Why  is  potassium  permanganate  used  in  this  method? 
Write  the  equation  to  represent  the  first  stage. 

■KCN  +  KMnO,  +  KOH  +  H?0  = 

PROPERTIES  OF   UREA. 

Melting-point. — Determine  the  melting-point  of  the  urea  fur- 
nished by  the  laboratory;  of  that  obtained  from  the  urine  and  of  that 
prepared  synthetically.     How  do  the  results  compare? 

The  determination  is  made  as  follows:  Heat  a  piece  of  soft 
glass  tubing  in  a  blast-lamp  and  when  thoroughly  softened  draw 
slowly  apart.  The  narrow  tube  thus  obtained  is  cut  up  into  pieces 
about  five  inches  long;  each  of  these  fused  in  the  middle  yields 
two  tubes  which  are  used  in  making  the  determination.  Fill  one  of 
these  tubes,  sealed  at  one  end,  to  a  height  of  ^i—Yz  inch  with  the 
substance,  then  fasten  this  by  means  of  a  small  rubber  band  cut  from 
a  piece  of  rubber  tubing,  to  a  thermometer  so  that  the  sealed  end  of 
the  tube  is  on  a  level  with  the  end  of  the  bulb  of  the  thermometer 
Suspend  the  thermometer  with  the  attached  tube  in  a  beaker  of 
about  100  c.c.  capacity,  containing  enough  sulphuric  acid  to  clear  the 
substance  in  the  tube  as  well  as  the  bulb  of  the  thermometer.  Now 
heat  with  a  small  flame,  stirring  constantly,  till  the  substance  begins 
to  melt.     This  is  the  melting-point;  note  the  temperature. 

Taste  a  specimen  of  pure  urea?    What  is  it  like? 

Examine  the  crystals  of  urea  under  the  microscope;  recrysta  llize , 
if  necessary.     Sketch  the  form  of  the  crystals  observed. 

Test  the  solubility  of  the  urea  in  water,  alcohol  and  ether. 


132  PHYSIOLOGICAL  CHEMISTRY. 

SALTS  OF  UREA. 

Urea  nitrate,  CO(NH2)2  .  HN03. — To  about  %  c.c.  of  concentrated 
urea  solution  or  to  some  urine  concentrated  to  about  one-third,  add 
an  excess  of  strong"  nitric  acid  free  from  nitrous  acid.  If  the  pre- 
cipitate which  forms  does  not  show  distinct  crystals,  redissolve  by 
aid  of  heat  and  pour  the  contents  of  the  test-tube  out  into  a  watch- 
glass.  Examine  the  crystalline  form  under  the  microscope  and  sketch 
the  same.  Test  the  solubility  of  the  crystals  in  water,  alcohol  and 
ether. 

Observe  the  formation  of  the  crystals  directly  under  the  micro- 
scope, as  follows:  Place  on  a  slide  a  drop  or  so  of  the  urea  solution, 
cover  and  apply  a  drop  of  nitric  acid  at  the  edge. 

Urea  nitrate  obtained  on  decomposition  of  adenin  and  brom- 
hypoxanthin  does  not  crystallize  in  six-sided  plates  (Kriiger). 

Urea  oxalate,  2CO(NH2)2 .  H2C204. — To  about  y2  c.c.  of  concen- 
trated urea  solution  add  some  concentrated  oxalic  acid  solution. 
Redissolve  the  precipitate  which  forms  by  the  application  of  gentle 
heat  and  pour  the  solution  into  a  watch-glass.  Study  and  sketch  the 
form  of  the  crystals  as  observed  under  the  microscope.  Test  the 
solubility  the  same  as  of  nitrate. 

Observe  the  formation  of  the  crystals  directly  under  the  micro- 
scope in  the  manner  given  for  the  nitrate. 

MERCURIC  NITRATE   COMPOUNDS. 

*1.—  2CO(NH2)2  .  Hg(N03)2  .  HgO.— A  compound  having 
this  composition  is  obtained  when  a  nitric  acid  solution  of 
mercuric  nitrate  is  added  to  a  moderately  dilute  solution  of 
urea  nitrate.  Examine  the  crusts  which  form  on  standing 
and  sketch  the  crystals. 

*2.—  2CO(NH2)2 .  Hg(N03)2.  2HgO.— This  is  formed  when 
mercuric  nitrate  is  added  to  a  urea  solution  as  long  as  a 
precipitate  forms  and  this  then  set  aside  for  some  time  at 
a  temperature  of  40 — 50°.  Study  and  sketch  the  crystals 
which  result. 

3.— 2CO(NH2)2  .  Hg(N03)2  .  3HgO.— A  compound  possessing  this 
formula  is  formed  when   a  faintly  acid,  approximately  7  per  cent. 


URINE.  133 

solution  of  mercuric  nitrate  is  added  to  a  2  per  cent,  solution  of  urea. 
Note  that  the  flocculent  precipitate  which  forms  becomes  granular 
on  standing.  One  of  the  methods  for  the  quantitative  estimation  of 
urea  depends  upon  the  production  of  this  precipitate. 

Treat  a  portion  of  the  precipitate  with  nitric  acid.  Note  the 
effect. 

Test  another  portion  with  salt  solution.  Observe  the  result  and 
explain. 

Write  the  equation  showing  the  formation  of  this  precipitate. 

Calculate  the  ratio  existing  between  the  molecular  weight  of 
urea  and  that  of  mercuric  oxide  from  the  composition  of  the  above 
precipitate. 

REACTIONS. 

Furfural  test. — Place  a  few  crystals  of  urea  in  a  porcelain  dish,, 
add  1 — 2  drops  of  a  concentrated  aqueous  solution  of  f urfurol  and  1 — 2 
drops  of  concentrated  hydrochloric  acid — a  faint  yellow  color  ap- 
pears which  in  a  few  minutes  changes  to  a  splendid  purple.  Inter- 
mediate tints  of  green,  blue  and  violet  may  precede  (Schiff ).  This 
test  is  also  given  by  allantoin  but  not  by  uric  acid. 

Benzoyl  chloride  test. — To  about  J4  c.c.  of  a  concentrated  aqueous 
solution  of  urea,  add  about  2  c.c.  of  sodium  hydrate  solution  and  yi 
c.c.  of  benzoyl  chloride,  then  close  the  tube  with  a  stopper  and  shake 
thoroughly.  The  tube  becomes  hot  and  benzoyl  urea,  CO(NH.COC6H5)2, 
forms.  Test  a  specimen  of  urine  (about  2  per  cent,  urea)  in  this  way. 
Note  the  result  and  explain.  Write  the  equation  representing  the 
reaction  between  urea  and  benzoyl  chloride  (COC1 .  C6H5). 

This  test  applied  to  urine  which  contains  much  ammonia  will 
give  a  precipitate  of  benzamide. 

Bloxam's  test. — Acidulate  a  few  crystals  with  hydrochloric  acid 
and  evaporate  in  a  dish  to  dryness,  then  heat  till  white  vapors  (biuret) 
cease  to  be  given  off.  Cool  and  test  according  to  directions  given 
under  Exp.  d,  p  135. 

DECOMPOSITIONS. 

1. — Into  ammonia  and  cyanate. — Urea,  as  shown,  is  prepared  syn- 
thetically from  ammonium  cyanate  and  on  decomposition  it  readily 
yields  the  compounds  from  which  it  is  formed.  Evaporate  to  dryness 
on  the  water-bath  a  few  c.c.  of  a  solution  of  urea  to  which  some  silver 
nitrate  has  been  added — silver  cyanate  and  ammonium  nitrate  form. 


134  PHYSIOLOGICAL  CHEMISTRY. 

Test  a  portion  of  the  residue  for  ammonium  by  warming  with  potas- 
sium hydrate.  Test  another  portion  for  silver  cyanate  as  follows: 
Treat  the  residue  with  cold  water  and  filter— silver  cyanate  is  spar- 
ingly soluble  in  water  and  hence  remains  on  the  filter.  To  prove  that 
this  residue  consists  of  silver  cyanate,  (1)  to  a  portion  add  ammonium 
hydrate.  What  is  the  result?  (2)  To  another  portion  add  dilute  nitric 
acid — note  the  effervescence.  What  is  it  due  to?  Write  an  equation 
to  represent  the  decomposition  of  urea  in  the  presence  of  silver 
nitrate,  also  an  equation  showing  the  action  of  nitric  acid  on  silver 
cyanate. 

2.— Into  Biuret,  C2H5N,02. — Heat  some  urea  in  a  test-tube  till  it 
melts  and  keep  it  at  that  temperature  until  gas  bubbles  are  freely 
given  off  from  the  fused  mass.  Test  the  odor  of  the  gas  evolved.  What 
is  it?  Now  set  the  tube  aside  to  cool,  then  dissolve  in  a  little  water 
and  test  as  follows  for  biuret:  To  the  aqueous  solution  add  some 
potassium  hydrate,  then  a  drop  or  less  of  a  dilute  copper  sulphate 
solution.  Note  the  bright  pink  color  [Biuret  reaction).  What  is  the 
result  when  more  copper  sulphate  is  added?  The  conversion  of  urea 
into  biuret  can  best  be  represented  thus: 


CO<NH2  CO<NH2 

CO<NH2  CO<NH2 


What  compounds  heretofore  studied  give  a  biuret  reac- 
tion? Biuret  unites  with  metals,  as  potassium,  mercury, 
copper,  nickel  and  cobalt.  With  copper  and  alkali  it  gives 
the  violet  or  red  color,  known  as  the  biuret  reaction.  If 
nickel  is  substituted  in  this  test  for  copper  a  yellow  or 
orange  color  results.  The  biuret  reaction  is  given  by  sub- 
stances containing  two  amido-carbonyl  groups  ( — CO  .  NH2)2, 
united  to  a  C  or  N  atom,  or  to  a  — CO  .  NH  group,  or  directly 
to  one  another,  as  in  oxamide.  The  proteid  molecule  con- 
tains probably  diamides,  though  not  necessarily  the  biuret 
group.     (Schiff). 

3. — Into  cyanuric  acid,  C3H3N3O3. — Heat  some  urea,  as  just  given 
under  biuret,  till  gas  ceases  to  be  evolved  and  the  contents  solidify 
to  a  white  chalky  mass.     Test  this  for  cyanuric  acid  as  follows: 


URINE.  135 

(a).  Insoluble  in  cold,  soluble  in  hot  water  from  which  it  recrys- 
tallizes  in  prisms. 

(6).  Dissolve  a  portion  of  the  residue  in  cold  concentrated 
sodium  hydrate,  then  heat.  The  sodium  salt  which  has  formed 
recrystallizes  in  needles  when  its  solution  is  heated. 

(c).  Dissolve  a  portion  of  the  residue  in  boiling"  water  and  add 
this  to  a  dilute  solution  of  ammonium  cupric  sulphate — a  beautiful 
violet  precipitate  results.  Avoid  excess  of  ammonium  hydrate  or 
of  copper  sulphate. 

(d).  Dissolve  a  portion  in  a  few  drops  of  ammonium  hydrate;  to 
one-half  of  the  solution  add  a  drop  of  barium  chloride  solution — a 
crystalline  precipitate  forms;  to  the  remainder  add  a  drop  of  a  weak 
copper  sulphate  solution — a  violet,  crystalline  precipitate  is  produced. 

/NH 
co    NH-2  CO 

UU<NH2  I 

NH 

co     NH2      _       1/  +3NH3. 

bU<NH2      -     co 


CO< 


NH2  \/ 

NH.2  CO 


NH 


4. — Boil  for  some  time  an  aqueous  solution  of  urea  in  a  test-tube 
in  the  mouth  of  which  is  placed  a  moist  red  litmus  paper.  What  is 
the  result  and  to  what  is  it  due?  Write  the  equation  representing 
the  decomposition  of  urea  into  ammonia  and  carbonic  acid. 

5. — Heat  some  urea  in  a  test-tube  with  potassium  hydrate.  Ob- 
serve the  odor,  and  when  the  tube  is  cold  acidulate  with  dilute  hydro- 
chloric acid — note  the  effervescence.     To  what  is  it  due? 

6.— To  a  few  c.c.  of  concentrated  urea  solution  in  a  test-tube  add 
some  nitrous  acid,  or  about  2  c.c.  of  strongly  colored  nitric  acid. 
What  is  the  result?  Write  an  equation  showing  the  action  of  nitrous 
acid  on  urea,  with  carbonic  acid,  nitrogen  and  water  as  resulting 
products. 

7.— To  some  urea  solution  (or  urine)  add  sodium  hypobromite  or 
hypochlorite.  Note  the  effect.  The  reaction  is  represented  by  the 
equation: 

CO(NH2)2  +  3  NaBrO  =  3  NaBr  -f  CO2  +  N2  +  2  H20. 

8. — To  about  2  c.c.  of  permanganate  of  potash  solution,  add  some 

10 


136  PHYSIOLOGICAL    CHEMISTRY. 

concentrated  urea  solution  and  then  about  Yz  c.c.  of  concentrated 
sulphuric  acid.     Note  the  effervescence. 

2  CO(NB2)2  +  Mn207  =  2  C02  +  N2  +  2  NH3  +  H20  +  2  MnOo. 

Write  this  equation  with  the  symbol  of  potassium  permanganate 
instead  of  Mn20,. 

9. — Set  aside  some  urine  for  several  days.  Then  test  the  reac- 
tion; what  is  it,  and  to  what  is  it  due?  What  is  the  cause  of  this 
decomposition?  To  a  portion  add  some  dilute  acid — observe  the  effer- 
vescence. 

Write  the  equation  showing  the  decomposition  of  urea  into 
ammonium  carbonate. 

Allow  the  litmus  paper  which  was  used  to  test  the  reaction  to 
dry  in  the  air.  Notice  that  the  original  color  returns.  The  color  was 
due  to  volatile  alkali — ammonia. 

Immerse  a  piece  of  red  litmus  paper  in  sodium  hydrate  solution 
and  then  set  aside  to  dry.  Notice  that  the  change  in  color  remains 
permanent — due  to  fixed  alkali. 

* Detection  of  urea  in  liquids  other  than  urine. — The  method 
as  given  in  Exp.  1,  p.  129,  for  the  isolation  of  urea  from 
urine  can  be  employed  for  the  detection  of  urea  in 
gastric  juice,  faeces,  blood,  pus,  etc.,  in  pathological  con- 
ditions. To  the  material  add  3 — 4  volumes  of  alcohol,  mix 
and  set  aside  for  24  hours,  then  filter  and  concentrate  to  a 
small  volume.  To  this  syrupy  liquid  add  HN03  and  exam- 
ine for  crystals  of  urea  nitrate.  To  the  crystals  also  apply 
the  biuret,  furfurol  and  nitrous  acid  tests. 

Ammonia. 

Normal  urine  always  contains  some  ammonia,  which  is 
not  free  but  combined  as  a  salt, — chloride,  sulphate  or 
phosphate.  About  0. 7  g.  of  ammonia  is  excreted  daily  by  an 
adult.  Of  the  total  waste  nitrogen  from  2 — 5  per  cent,  may 
appear  in  the  urine  as  ammonia,  which  therefore  may  be 
looked  upon  as  second  in  importance  as  a  carrier  of  waste 
nitrogen.     As  stated  under  urea,  ammonium  acetate  or  any 


URINE.  137 

other  ammonium  salt  of  an  organic  acid,  when  administered, 
ii  oxidized  in  the  body  to  ammonium  carbonate  which  in 
turn  is  converted  into  urea.  When  the  ammonia  is  united 
with  a  strong  mineral  acid,  as  chloride,  sulphate  or  phos- 
phate, it  passes  through  the  body  unchanged  and  will 
appear  as  such  in  the  urine.  Because  of  this  union  with  a 
strong  acid  it  cannot  be  converted  into  urea.  Again,  if 
mineral  acids  are  administered,  as  hydrochloric  acid,  the 
amount  of  the  corresponding  ammonium  salt  in  the  urine 
will  be  increased.  The  acid  combines  with  ammonia  and 
hence  this  escapes  conversion  into  urea. 

The  presence  of  ammonia  in  the  urine  is  explained  by 
these  facts.  The  sulphur  or  phosphorus  of  the  food  and 
tissues  are  oxidized  in  the  body  to  sulphuric  and  phosphoric 
acids  respectively.  A  part  of  these  acids  unite  with 
ammonia  and  the  result  is  the  same  as  if  these  acids  were 
administered.  For  this  reason  the  amount  of  ammonia  in 
the  urine  after  a  meat  diet  is  greater  than  after  a  vegetable 
diet;  is  greater  in  the  urine  of  carnivorous  as  compared 
with  herbivorous  animals.  The  urine  in  starvation  will  for 
like  reason  contain  ammonium  salts.  Some  ammonium  salts 
may  be  introduced  with  the  food,  as  in  the  case  of 
cheese,  or  may  be  formed  as  a  result  of  putrefaction  in  the 
intestines. 

An  increase  in  ammonia  may  be  expected  in  diseases 
where  the  proteins  of  the  body  are  being  unduly  disinte- 
grated. This  is  the  case  in  fever,  and  in  diabetes  mellitus. 
Since  urea  is  made  by  the  liver  out  of  ammonium  carbonate 
or  carbamate,  it  follows  that  in  structural  diseases  of  the 
liver  ammonia  in  the  urine  may  be  greatly  increased. 

Normal  urine,  which  is  acid  in  reaction  when  passed, 
on  standing  undergoes  ammoniacal  fermentation  due  to  the 
introduction  of  various  kinds  of  bacteria.  These  organisms 
may  be  introduced  into  the  bladder  as  a  result  of  injury 
or  from  the  use  of  unsterilized  catheters,  and  hence  this 
fermentative  change  may  be  going  on  in  the  urine  at  the 


138  PHYSIOLOGICAL   CHEMISTRY. 

time  of  passage.  Such  urine  will  be  cloudy,  will  possess  an 
ammoniacal  odor  and  with  acids  will  effervesce  because  of 
the  presence  of  ammonium  carbonate.  This  compound 
results  from  the  hydration  of  the  urea  (Exp.  9,  p.  136). 
The  reaction  of  such  urine  is  alkaline,  due  to  volatile  alkali 
and  should  be  distinguished  from  that  due  to  fixed  alkali, 
as  sodium  carbonate. 


NH— C— NH 
Uric  Acid,  c5H4N403,     =        CO 

NH— C     CO  . 

I       I 
CO-NH 


Although  uric  acid  is  present  in  the  normal  urine  in 
comparatively  small  quantity,  on  an  average  0.7  g.  per 
day,  it  nevertheless  constitutes  one  of  the  most  important 
constituents  of  urine,  especially  in  disease.  As  a  nitrogen 
carrier  it  ranks  third,  since  it  contains  1  to  3  per  cent,  of 
the  total  waste  nitrogen. 

Although  uric  acid,  as  can  be  seen  from  the  structural 
formula,  contains  two  urea  groups  and  can,  as  a  matter  of 
fact  on  decomposition,  yield  two  molecules  of  urea  it  does 
not  follow  that  uric  acid  is  an  antecedent  of  urea.  When 
fed  to  mammals  it  is  broken  up  in  the  body  and  excreted  as 
urea;  and  it  is  possible  for  some  of  the  uric  acid  made  in 
the  body  to  undergo  similar  conversion  into  urea.  On  the 
other  hand,  administration  of  urea  or  of  ammonium  salts 
to  birds  is  said  to  increase  the  amount  of  uric  acid  excreted. 

It  has  been  shown  that  urea  is  the  final  waste  product 
of  proteins.  These  may  be  present  in  the  circulating  fluids, 
or  in  the  protoplasm  of  the  cells.  The  nuclei  of  cells, 
however,  have  a  different  composition  from  protoplasm. 
They  contain  complex  proteids,  the  so-called  nucleo-pro- 
teids,  which  on  decomposition  yield  the  nucleins.     It  has 


URINE.  139 

been  known  for  some  years  that  nuclein  on  decomposition 
with  acids  or  alkalis  yields  the  xanthin  bases.  As  seen 
from  the  formulas  xanthin  is  closely  related  to  uric  acid, 
the  latter  containing  one  atom  more  of  oxygen. 

Xanthin,  C5H4N402. 
Uric  Acid,  C5H4N403- 

The  source  of  the  xanthin  is  clearly  nuclein,  and  the 
relation  of   these  bases  to  uric  acid  rendered  it  probable 
that  the  source  of   uric  acid  was  likewise  nuclein.     This 
has  been  demonstrated  by  the  researches  of  Horbaczewski. 
When  tissue  rich  in  nuclein  is  first  partially  oxidized,  then 
decomposed  it  yields  uric  acid.     Without  oxidation  only  the 
xanthin  bases   would   be  formed.      All  tissues  containing 
nucleated  cells  can  be  thus  made  to  yield  uric  acid.     The 
more  nucleated  cells  present,  the  greater  the  yield  (as  from 
spleen);  the  fewer  nucleated  cells  present,  (tendons,  etc.), 
the  smaller  will  be  the  yield  of  uric  acid.     It  is  evident, 
therefore,  that   uric  acid  is  to  be  regarded  as  a  specific 
waste   product  of  nuclein  decomposition.      Its  relation  to 
nucleated  cells  is  seen  in  leukamia,  which  is  characterized 
by  a  large  increase  in  the  number  of  white  blood  cells.     In 
the  urine  of  this  disease  xanthin  bases  and  uric  acid  are 
greatly   increased.      Again,    certain    chemical    substances 
(pilocarpin)  which  increase  leucocytes  increase  the  amount 
of   uric  acid,  whereas  others  (quinine,  atropine)  which  de- 
crease leucocytes  also  decrease  the  amount  of  uric  acid. 

In  birds,  reptiles,  amphibians,  etc.,  uric  acid  is  the 
chief  form  in  which  nitrogen  is  eliminated.  They  are  differ- 
ent from  mammals,  in  which  urea  is  the  chief  nitrogenous 
waste  product.  The  source  of  the  uric  acid  of  birds,  etc., 
is  not  necessarily  the  same  as  that  of  mammals.  This  is 
indicated  in  the  fact  mentioned  that  urea  and  ammonium 
salts  administered  to  birds  increase  the  amount  of  uric  acid 


140 


PHYSIOLOGICAL  CHEMISTRY. 


excreted.  Moreover,  after  complete  removal  of  the  liver 
from  geese  these  birds  excrete  large  amounts  of  ammonia 
and  lactic  acid.  It  is  probable,  therefore,  that  in  these 
animals  uric  acid  may  be  made  synthetically  in  the  liver 
out  of  ammonia  and  lactic  or  other  acids.  Some  uric  acid 
may  result,  as  in  mammals,  from  nuclein  decomposition; 
and  since  these  animals  have  nucleated  blood-cells  the 
amount  of  uric  acid  derived  from  this  source  may  be 
considerable. 

Uric  acid,  like  other  nitrogenous  constituents  of  the 
urine,  rises  and  falls  according  to  the  amount  of  protein 
matter  in  the  food.  This  is  well  illustrated  in  the  following 
analytical  results  obtained  by  Bunge. 


Urea. 


Creatinin. 


Uric  Acid. 


Meat  diet. . 
Bread  diet. 


67.2  g. 
20.6  " 


2.163  g. 
0.961  " 


1.398  g. 
0.253  " 


Fats  and  carbohydrates  in  the  food  or  in  the  tissues, 
owing  to  their  proteid  saving  action,  diminish  the  amount 
of  uric  acid  excreted  as  well  as  that  of  nitrogenous  pro- 
ducts in  general.  Excessive  muscular  exercise,  as  in 
prolonged  marches,  is  followed  by  an  increase  in  uric  acid. 
In  infants  and  children  the  uric  acid  excretion  is  relatively 
higher  than  in  adults. 

The  administration  of  pilocarpin  increases  leucocytes 
and  hence  increases  the  amount  of  uric  acid  eliminated. 
Antipyrin,  according  to  some,  increases  uric  acid,  whereas 
others  have  found  a  decrease.  Conflicting  opinions  exist 
as  to  the  effect  of  sodium  salicylate  but  the  preponderance 
of  evidence  goes  to  show  that  it  increases  uric  acid.  Alco- 
hol given  to  dogs  greatly  increases  the  amount  of  uric  acid. 
It  also  seems  to  increase  uric  acid  when  given  as  cham- 
pagne, but  not  when  given  as  whisky.  Glycerine  likewise 
causes  an  increased  excretion. 


URINE.  141 

Quinine,  in  small  doses,  greatly  diminishes  uric  acid. 
A  decrease  is  also  brought  about  by  atropine  and  by 
antifebrin. 

While  in  the  case  of  urea  the  chief  pathological  im- 
portance is  attached  to  a  decrease  in  the  excretion  of  that 
body,  the  reverse  is  true  of  uric  acid.  Uric  acid  and  urates 
acquire  their  chief  significance  when  present  in  excess,  and 
the  danger  lies  in  the  formation  of  deposits,  eventually  of 
calculi.  Some  persons  exhibit  a  marked  tendenc}^  to  uric 
acid  excretion  and  this  condition  is  designated  as  uric  acid 
diathesis,  or  lithemia.  Stimulants  as  alcohol,  coffee,  etc., 
which  experimentally  may  not  induce  increased  excretion 
of  uric  acid  in  animals  are  capable  of  doing  this  in  persons 
so  predisposed. 

The  most  marked  and  constant  increase  in  uric  acid  is 
met  with  in  leukamia.  In  this  disease  the  white  blood-cells 
are  greatly  increased.  The  amount  of  uric  acid  may  rise 
to  4,  5  or  even  6  g.  per  day.  Uric  acid  is  furthermore 
increased  in  fevers,  in  pernicious  anaemia,  arthritis  and  in 
certain  diseases  of  the  heart  and  lungs.  An  excess  of  uric 
acid  is  found  in  certain  nervous  disorders  as  neurasthenia, 
migraine,  epilepsy  and  chorea,  especially  after  the  attacks. 

In  gout  and  rheumatism  a  decrease  of  uric  acid  is  said 
to  occur,  due  as  is  supposed  to  the  retention  and  deposition 
of  uric  acid  and  urates  in  the  joints.  A  decrease  also  exists 
in  diabetes. 

Several  standards  have  been  employed  to  decide  when 
uric  acid  is  present  in  excess.  Thus,  by  some  the  absolute 
amount  of  uric  acid  excreted  in  24  hours  is  taken  as  the 
standard.  Since  a  person  normally  excretes  0.75  g.  per 
day,  1. 5  g.  would  therefore  be  considered  an  excess.  On  the 
other  hand  the  amount  of  urea  excreted  may  be  increased  at 
the  same  time  and  hence  the  ratio  between  urea  and  uric  acid 
will  be  the  same  as  in  normal  urine.  Thus  0.75  g.  uric  acid 
and  30  g.  urea  gives  a  ratio  of  1  to  40;  1.5  g.  of  uric  acid 
excreted  with  60  g.  of   urea  would  still  give  the  normal 


142  PHYSIOLOGICAL  CHEMISTRY. 

ratio.  Consequently  the  ratio  of  uric  acid  to  urea  (1  to  40) 
is  frequently  taken  as  the  standard  of  comparison. 
Another  standard  is  the  ratio  between  the  nitrogen  con- 
tained in  the  uric  acid  and  the  total  nitrogen  m  the  urine. 
Usually  the  normal  quotient  of  this  ratio  is  placed  at  50. 

Uric  acid  can  be  readily  prepared  (1)  from  the  excrement  of 
serpents,  (2)  from  guano,  (3)  from  uric  acid  calculi,  (4)  from  urine. 

*  Preparation  from  guano. — Boil  some  Peruvian  guano 
repeatedly  with  milk  of  lime  and  water  till  the  solution 
ceases  to  become  colored.  Then  extract  the  insoluble  resi- 
due with  boiling-  sodium  carbonate  till  the  filtrate  on 
addition  of  hydrochloric  acid  ceases  to  give  a  precipitate. 
The  combined  filtrates  are  treated  with  sodium  acetate  and 
then  hydrochloric  acid  added  to  a  distinct  acid  reaction. 
The  precipitate  which  consists  of  uric  acid  and  guanin,  is 
washed  and  then  boiled  with  moderately  dilute  hydrochloric 
acid  whereby  the  guanin  is  dissolved  while  the  uric  acid 
remains  behind. 

Preparation  from  the  urine.  1. — To  about  500  c.c.  of  normal 
filtered  urine,  free  from  albumin,  add  10 — 15  c.c.  of  concentrated 
hydrochloric  acid  and  set  aside  for  24—48  hours.  The  uric  acid 
deposits  in  strongly  colored  crystals.  Examine  the  crystals  under  the 
microscope  and  sketch  the  several  forms  observed.  The  deposit  can 
be  purified  by  dissolving  in  dilute  alkali,  then  decoloring  with  animal 
charcoal  and  finally  reprecipitating  the  uric  acid  with  hydrochloric 
acid. 

*2. — The  best  method  for  the  isolation  of  uric  acid, 
especially  when  the  amount  is  small,  is  Ludwig's  method, 
i.  e.  precipitation  with  magnesia  mixture  and  ammoniacal 
silver  nitrate.  The  precipitate  is  washed  with  ammonia 
water  then  decomposed  by  warming  with  potassium  sul- 
phide and  filtered.  The  filtrate  after  addition  of  hydro- 
chloric acid  is  concentrated  to  a  small  volume,  when  the 
uric  acid  crystallizes.     (See  Chapter  XI). 


URINE.  143 

*  Synthetic  preparation.  (Horbaczewski).  Heat  in  a 
test-tube,  in  a  small  flame,  0.1 — 0.3  g.  glycocoll  with  1 — 2  g. 
of  urea  till  the  fused  mass  becomes  solid.  Avoid  heating- 
above  220°.  The  brownish-yellow  mass  thus  obtained  can 
be  tested  for  uric  acid  by  applying  the  murexid  test 
(p.  145).  To  isolate  the  uric  acid  the  contents  of  several 
tubes  thus  treated  are  dissolved  in  boiling  water  with 
addition  of  some  ammonium  hydrate  and  the  filtrate  treated 
with  magnesia  mixture  and  ammoniacal  silver  nitrate;  from 
the  resulting  precipitate  uric  acid  is  obtained  as  given 
under  Ludwig's  method. 

The  synthetic  process  can  be  represented  by  the 
equation : 

CH2(NH2) .  COOH  +  3  CO(XH2)2  =  C5H4N403  +  3  NH3  +  2  H20. 

PROPERTIES  OP   URIC  ACID. 

Examine  under  the  microscope  the  form  of  the  crystals  of  the 
laboratory  specimen  and  of  that  obtained  form  the  urine. 

Place  a  few  crystals  on  a  slide,  add  a  drop  of  potassium  hydrate 
solution  and  watch  the  solution  of  the  crystals  through  the  micro- 
scope. When  dissolved  apply  a  drop  of  concentrated  acetic  acid  to 
the  edge  of  the  cover-glass,  and  again  examine.     What  is  the  result? 

Test  the  solubility  of  the  crystals  in  water,  alcohol,  ether, 
potassium  hydrate,  ammonium  hydrate,  hydrochloric  acid.  Uric  acid 
is  soluble  in  sodium  phosphate  and  is  reprecipitated  by  acids  (Wulff ). 
About  0.7  g.  of  uric  acid  may  be  held  in  solution  in  urine  by  the  urea 
present.  The  phosphates  may  still  further  increase  the  solubility. 
It  is  very  soluble  in  piperazin,  or  in  lysidin. 

SALTS  OF  URIC  ACID. 

8odium  acid  urate,  C5H3NaN403.— To  some  uric  acid  in  a  test- 
tube  add  water  and  boil,  then  add  sodium  hydrate,  drop  by  drop,  till 
it  dissolves.  To  the  solution  thus  obtained  add  sodium  bicarbonate, 
or  pass  carbonic  acid  gas  till  it  is  almost  neutral.  Set  aside  over 
night  to  crystallize.  Recrystallize,  if  necessary,  from  hot  water  and 
examine  the  crytals  under  the  microscope.  Sketch  a  few  of  the 
same.     What  is  the  formula  of  the  normal  sodium  urate? 


144  PHYSIOLOGICAL  CHEMISTRY. 

Potassium  acid  urate,  C5H3KN403,  is  prepared  in  the  same 
manner  as  already  given,  using"  however,  potassium  hydrate  instead 
of  sodium  hydrate.     Examine  and  sketch  the  crystalline  form. 

Ammonium  acid  urate,  C5H3(NH4)N403. — This  is  readily  prepared 
in  the  manner  already  given,  by  boiling  uric  acid  with  ammonium 
hydrate.  If  the  precipitate  fails  to  dissolve  and  the  solution  is 
strongly  ammoniacal,  add  water  till  complete  solution  results,  then 
set  aside  to  crystallize. 

Examine  the  crystals  and  compare  with  the  preceding. 

Does  the  normal  ammonium  urate  exist? 

Calcium  urate  can  be  prepared  in  a  similar  manner  as  the  pre- 
ceding by  adding  calcium  hydrate  to  the  boiling  mixture  till  the  uric 
acid  dissolves.     On  cooling  the  salt  crystallizes.     Examine  as  before. 

*Sulphate  of  uric  acid,  C5H4N403  -4  H2S04.  Add  uric 
acid  to  some  hot  concentrated  sulphuric  acid  as  long- 
as  it  dissolves.  On  cooling  large  transparent  crystals 
separate,  which  on  the  addition  of  water  decompose  into 
the  constituents. 

REACTIONS. 

1. — To  some  uric  acid  add  water,  boil,  then  add  ammonium 
hydrate,  drop  by  drop,  till  it  dissolves.  Dilute  with  an  equal  volume 
of  water,  add  hydrochloric  acid  to  acid  reaction  and  immediately 
after  add  a  solution  of  phosphotungstic  acid — a  bright  chocolate- 
brown  granular  precipitate  appears. 

2. — To  some  uric  acid  dissolved  as  above,  add  picric  acid — a  volu- 
minous yellow  precipitate  forms  (Jaffe). 

3. — To  uric  acid  dissolved  and  diluted  as  above,  add  ammoniacal 
silver  nitrate.  Note  the  result.  Now  add  a  little  of  a  solution  of  a 
neutral  salt  as  sodium  chloride,  ammonium  sulphate,  or  better  still 
magnesia  mixture — a  flocculent  or  gelatinous  precipitate  is  at  once 
thrown  down.  It  is  a  compound  of  uric  acid,  silver  and  the  base 
employed.     What  is  magnesia  mixture? 

i. — To  some  uric  acid  add  water,  boil,  and  then  add  a  few  drops  of 
sodium  hydrate  and  a  few  drops  of  Fehling's  solution.  On  heating 
the  white  cuprous  urate  is  thrown  down.  Allow  the  precipitate  to 
subside,  decant  the  supernatant  fluid  and  add  some  more  Fehling's 
solution,  then  boil  for  some  time.  The  red  cuprous  oxide  is  gradually 
formed.     (Compare  with  test  for  sugar  p.  19). 


URINE.  145 

This,  so-called  DrechseVs  reaction,  has  been  utilized  as  the  basis 
of  a  quantitative  method  of  separation  of  uric  acid  and  xanthin  bases 
(see  alloxuric  bodies). 

What  is  Fehling's  solution?    Write  the  formula  of  cuprous  oxide. 

5.— Dissolve  a  few  crystals  of  uric  acid  in  a  little  sodium  hydrate, 
then  pour  the  liquid  upon  a  filter  which  previously  has  been  moistened 
with  a  drop  of  silver  nitrate  solution— a  yellowish  to  a  brownish-black 
stain  indicates  reduced  silver.  This  is  a  very  delicate  test  and  is 
given  by  as  little  as  ^-^  mg.  of  uric  acid. 

6. — Mwrexid  test.— Place  a  minute  quantity  of  uric  acid  in  an 
evaporating  dish,  add  nitric  acid  and  evaporate  to  dryness  on  the 
water-bath.  A  yellowish  residue  results  which  on  contact  with  the 
vapors  of  ammonia  turns  to  a  beautiful  pink  or  red  (ammonium  pur- 
purate  or  murexid).  Now  add  a  drop  of  potassium  hydrate.  Note 
the  change  in  color  and  the  fact  that  this  disappears  in  a  short  time 
on  standing — distinction  from  the  xanthin  compound,  which  in  addi- 
tion has  more  of  a  red  color. 

The  change  that  takes  place  in  the  above  reaction  is  as  follows: 
The  uric  acid  is  oxidized  by  the  nitric  acid  to  alloxantin  which  is  a 

combination  of  alloxan  and  dialuric  acid  (  CO<S§— S2>CH — OH  j    . 

I  Nix — GO  j 

Addition  of  ammonia  converts  the  latter  into  dialuramide,  (uramil) 

CO<S5"£q>CH— NH2,  which  with  alloxan  yields    purpuric   acid. 

Excess  of   ammonia  produces  ammonium  purpurate  or  murexid  (see 
formula  p.  152). 

Why  is  it  called  murexide?  Is  purpuric  acid  known  in  the  free 
state? 

7. — Heat  sharply  some  uric  acid  in  a  test-tube.  It  decomposes 
into  ammonia,  hydrocyanic  acid  (recognized  by  its  peach-blossom 
odor),  urea  and  cyanuric  acid. 


DECOMPOSITIONS. 

Uric  acid  on  oxidation  may  yield  three  distinct  groups 
of  products. 

1. — In  cold  acid  solution  it  yields  urea  and  alloxan — 
the  alloxan  group. 


146 


PHYSIOLOGICAL  CHEMISTRY. 


2. — In  warm  acid  solution  it  yields  parabanic  acid — the 
parabanic  group. 

3. — In  neutral  or  alkaline  solution  it  yields  allantoin 
— the  allantoin  group. 

An  inspection  of  the  structural  formula  of  uric  acid 
reveals  the  presence  of  two  urea  groups;  or  we  may  say 
one  urea  and  one  alloxan  group.  The  first  cleavage  of  the 
uric  acid  molecule  results  in  the  formation  of  these  two 
molecules: 


NH— C 
CO 
NH— C 


-NH 


CO 


CO — NH 

Uric  acid. 


NH2 

I 
+  H2O  +  O  =     CO 

I 

NH2 
Urea. 


CO— NH 

I         I 
+     CO    CO 

I     I 

CO— NH 

Alloxan. 


On  further  oxidation  alloxan  yields  carbonic  acid  and 
parabanic  acid: 

CO— NH  CO— NH 

CO    CO     +  o  =  co2  +  CO    . 

CO— NH  CO— NH 

Parabanic  acid. 

Parabanic  acid   takes   up  the  elements  of   water  and 
yields  oxaluric  acid: 


CO— NH 

I 
CO 

^O— NH 


CO- 


+  H20  = 


-NH 
CO 


CO.  OH        NH2 
Oxaluric  acid. 


Oxaluric  acid  on  further  hydration  yields  oxalic  acid 
and  urea: 


CO- 


— NH 

I 
CO 

CO.OH        NH2 


+  H20  = 


CO.OH 


CO.OH 


NH2 

+    CO 

I 
NH, 


URINE.  147 

On  further  decomposition  the  oxalic  acid  yields  C02  and 
H20;  the  urea  likewise  splits  up  into  C02  and  NH3.  The 
uric  acid  molecule  can  therefore  be  easily  oxidized  to  the 
simple  inorganic  compounds,  C02,  NH3  and  H20. 

Of  the  above  mentioned  decomposition  products  of  uric 
acid,  alloxan  (alloxantin)  and  parabanic  acid  are  not  found 
in  the  urine.  Allantoin  and  oxaluric  acid  are  present  in 
the  urine  in  small  amount. 

The  fact  that  oxaluric  acid  and  allantoin,  oxidation 
products  of  uric  acid,  are  present  in  urine  indicates  clearly 
that  a  portion  of  the  uric  acid  formed  from  nuclein  is  oxi- 
dized in  the  body,  and  that  possibly  onty  a  small  amount 
escapes  conversion  and  appears  in  the  urine. 

Oxaluric  acid  exists  in  traces  as  an  ammonium  salt  in 
the  urine.  It  is  not  precipitated  by  calcium  chloride  and 
ammonia.  On  heating-  with  water,  acids  or  alkalis  it 
readily  undergoes  hydration.  It  forms  a  white  crystalline 
powder  and  yields  characteristic  ammonium  and  silver 
salts. 

Allantoin  was  first  met  with  in  the  allantoic  fluid  of  the 
cow.  It  is  found  in  the  urine  of  infants  during  the  first 
week  after  birth.  It  is  present  in  the  urine  during  preg- 
nancy and  is  probably  present,  though  in  minute  quantity, 
in  the  urine  of  adults.  It  is  present  in  the  urine  of  suckling 
calves,  and  at  times  in  the  urine  of  other  animals.  It  has 
been  found  in  leukamic  blood  and  in  ascitic  fluids.  When 
uric  acid  is  fed  to  dogs  allantoin  is  increased.  It  is  also 
increased  after  administration  of  tannic  acid,  and  of  di- 
amides. 

Allantoin  crystallizes  in  bright,  transparent  crystals 
which  are  but  slightly  soluble  in  cold  water.  In  many  of 
its  reactions  it  resembles  urea.  The  quantitative  deter- 
mination of  urea  with  mercuric  nitrate,  for  this  reason, 
will  include  allantoin. 


148  PHYSIOLOGICAL,  CHEMISTRY. 

Alloxantin,  C8H6N408  -f  2  H20. 

To  5  g.  of  uric  acid  in  a  small  Erlenmeyer  flask  add  10  c.c.  of 
concentrated  hydrochloric  acid  (sp.  gr.  1.19)  and  10  c.c.  of  water. 
Now  add  very  slowly  and  in  small  portions  1.5  g.  of  finely  pulverized 
potassium  chlorate.  No  chlorine  or  carbonic  acid  should  be  evolved. 
Nearly  all  the  uric  acid  passes  into  solution  as  urea  and  alloxan, 
C4H2N204,  which  on  reduction  can  readily  be  converted  into  alloxan- 
tin. For  this  purpose  dilute  the  liquid  with  an  equal  volume  of  water, 
filter  off  the  unchanged  uric  acid,  and  through,  the  filtrate,  returned 
to  the  flask,  pass  hydrogen  sulphide  as  long  as  a  precipitate  continues 
to  form.  Alloxantin  is  thrown  .out  of  solution  mixed  with  free  sul- 
phur. Set  the  flask  aside  over  night  in  the  cold.  Filter,  and  from 
the  precipitate  extract  the  alloxantin  by  heating  it  several  times 
with  boiling  water,  and  filtering.  Alloxantin  crystallizes  from  the 
filtrate  on  cooling  in  colorless  prismatic  crystals.  Filter  off  the  crys- 
tals and  dry  between  filter-paper. 

*The  filtrate  from  the  sulphur-alloxantin  precipitate  may  contain 
alloxantin.  To  recover  this  evaporate  in  vacuo,  extract  with  alco- 
hol, wash  the  insoluble  portion  in  cold  water  and  finally  crystallize 
from  hot  water  (Kriiger). 

Write  out  the  following  equations  which  represent  the  two 
stages  in  the  process. 

C5H4N403  +  O  +  H20  = 
2  C4H2N,04  +  H2S  = 

Alloxantin  has  been  found  to  be  a  cleavage  product  of  the  con- 
vicin  of  vetch  (plant  uric  acid).  It  is  of  interest  to  note  that  the 
latter  yields,  therefore,  a  typical  murexid  test. 

1. — Examine  the  crystals  under  the  microscope  and  sketch  the 
form.     (See  Roscoe  and  Schorlemmer,  Vol.  Ill,  Part  2). 

2. — Place  a  few  crystals  in  an  evaporating  dish,  and  crush;  then 
add  a  drop  of  concentrated  nitric  acid  and  evaporate  gently  over  a 
flame,  or  on  a  water-bath.  Moisten  the  residue  with  a  little  water 
and  add  a  drop  of  ammonium  hydrate— murexid  test. 

3. — To  a  few  crystals  of  alloxantin  in  a  porcelain  dish  add  a  drop 
of  ammonium  hydrate.  Note  the  results.  Then  add  a  drop  of  potas- 
sium hydrate  and  observe  the  change,  if  any.  To  what  is  this  due? 
Alloxantin  on  heating  with  ammonia  yields  first  uramil,  then 
murexid. 


URINE.  149 

4. — Dissolve  a  few  crystals  of  alloxantin  in  boiling-  water  and  add 
some  barium  hydrate  solution.  A  violet-blue  precipitate  forms  which 
on  heating  becomes  white  (due  to  formation  of  barium  alloxantate 
and  dialurate). 

5. — To  some  alloxantin  dissolved  as  above  add  a  little  ferrous 
sulphate,  then  some  ammonium  hydrate.  A  blue  color  results 
(Kruger). 

6. — To  an  aqueous  solution  of  alloxantin  add  some  ammoniacal 

silver  nitrate.     The  silver  is  reduced,  slowly  in  the  cold,  rapidly  on 

gentle  heating. 

NH— CO 

I  I 

Alloxan,    c4H,N,04,     =      CO    CO 

NH— CO. 

*To  the  powdered  alloxantin  prepared  as  just  given 
(about  2  g.)  add  about  4  c.c.  of  fuming-  nitric  acid  (1.50  sp.  gr.) 
and  2.5  c.c.  of  concentrated  nitric  acid  (1.42  sp.  gr.).  Rub 
up  in  a  mortar  or  dish,  then  set  aside  in  a  stoppered  test- 
tube  for  several  days  till  a  specimen  of  the  crystals  taken 
out  dissolves  readily  and  completely  in  water,  which  occurs 
as  soon  all  the  alloxantin  has  been  converted  into  alloxan. 
When  this  is  the  case  transfer  the  crystals  to  a  porous 
porcelain  plate  (or  asbestos  filter)  to  dry,  then  place  in  a 
porcelain  dish  and  heat  on  the  water-bath,  stirring  con- 
stantly, till  the  odor  of  nitric  acid  disappears.  Recrystal- 
lize  from  a  very  small  amount  of  hot  water. 

The  reaction  is  represented  by  the  equation: 

CSH(;NA  +  HNO:j  =  2  C4H2NA  +  HN02  4-  H20. 

1. — Study  and  sketch  the  crystalline  form  of  alloxan. 
How  do  the  crystals  behave  on  exposure  to  air? 

2. — On  boiMng  with  barium  hydrate  it  decomposes  into 
C02H 
mesoxalic   acid,  CO    ,  and  urea.     Write  out  the  equation, 

6o2h 


150  PHYSIOLOGICAL    CHEMISTRY. 

employing-  graphic  formulae.  On  warming  with  nitric  acid 
it  is  oxidized  to  parabanic  acid.  This,  by  the  action  of 
alkalis,  yields  oxaluric  acid,  which  in  turn  decomposes 
into  oxalic  acid  and  urea. 

Write  out  the  equations  representing  these  three 
changes.     (See  p.  146). 

4. — Formation  of  murexid:  Dissolve  a  few  crystals  of 
alloxan  in  a  little  water  in  a  dish,  evaporate  to  dryness 
and  add  ammonium  hydrate. 

5. — To  an  aqueous  solution  of  alloxan  add  excess  of 
baryta  water— a  white  precipitate  of  barium  alloxantate 
forms. 

NH-CH-NH 

Allantoin,  c4H6N403,     =      CO 

NH— CO     CO 
NH2. 

To  4  g.  of  uric  acid,  stirred  up  in  about  100  c.c.  of  water,  and 
warmed,  add  sodium  hydrate  till  it  dissolves,  and  when  cold  add 
gradually  3  g.  of  powdered  potassium  permanganate.  As  soon  as  all 
the  permanganate  has  been  added  and  dissolved,  filter.  Acidulate 
the  filtrate  with  acetic  acid  as  soon  as  possible,  then  set  aside  in  a 
cold  place  over  night.  Filter  off  the  crystals  which  form  and  wash 
with  water;  combine  the  wash-water  and  filtrate,  concentrate  on  the 
water  bath  to  a  small  volume,  then  set  aside  overnight.  The  crystals 
that  form  can  be  combined  with  those  previously  obtained  and  recrys- 
tallized  from  a  small  amount  of  hot  water. 

The  oxidation  takes  place  quantitatively  according  to  the 
equation: 

C5H4N403  +  H20  +  O  =  C02  +  C.HeNA- 

Write  out  the  following  equation  in  which  potassium  perman- 
ganate is  used  as  above. 

C5H4N403  +  H20  +  KMn04  = 

1. — Examine  and  sketch  the  crystalline  form. 

2. — Test  the  solubility  in  water:  also  test  the  reaction. 


URINE.  151 

3. — To  an  aqueous  allantoin  solution  add  a  drop  or  two  of  silver 
nitrate  solution.  No  precipitate  forms;  then  add  a  drop  of  dilute 
ammonium  hydrate  when  a  white  flocculent  precipitate  of  allantoin- 
silver,  C4H5AgN403,  results.  Test  the  solubility  in  ammonium  hydrate 
and  in  nitric  acid;  also  examine  the  precipitate  under  the  microscope 
— it  appears  as  droplets. 

4. — To  some  allantoin  solution  add  mercuric  nitrate  when  a  white 
flocculent  precipitate  forms.  Compare  with  the  behavior  of  urea 
(p.  132).  To  the  precipitate  add  ammonium  hydrate  and  warm.  Note 
the  change  and  explain. 

5. — Allantoin,  like  urea,  gives  the  furfurol  test  (p.  133),  except 
that  it  comes  slower  and  is  less  intense. 

6. — It  does  not  yield  the  murexid  test. 

7. — On  prolonged  boiling  with  Fehling's  solution  it  yields  cuprous 
oxide.     Compare  with  uric  acid  (p.  144). 

8. — Dissolve  some  allantoin  in  a  little  potassium  hydrate  and 
divide  the  solution  into  two  portions.  To  one  add  acetic  acid — the 
allantoin  is  precipitated.  Set  the  other  portion  aside  for  several 
days,  then  add  acetic  acid — no  precipitate  forms.  The  allantoin  has 
been  converted  into  allantoic  acid,  C4H8N404. 

9. — On  heating  with  acids,  allantoin  yields  allanturic  acid, 
C3H4N,03  ,  and  urea, 

C4H,N40,  +  H20  =  C3H4N,03  +  CO(NH2)2. 

10. — Boil  some  allantoin  with  concentrated  sodium  hydrate  till 
ammonia  vapors  are  given  off  (what  does  this  indicate?);  then  acidu- 
late with  acetic  acid,  and  add  a  few  drops  of  calcium  chloride  solu- 
tion. Test  the  precipitate  with  acetic  and  hydrochloric  acids.  What 
is  it?  Alkalis  yield  the  same  products  as  acids,  but  on  prolonged 
heating  the  allanturic  acid  decomposes  into  hydantoic  and  parabanic 
acids.  The  parabanic  acid  in  turn  is  decomposed  into  oxalic  acid  and 
urea. 

2C8HiN208     =     C3H8N2Os    +    C8H2N2Os. 

Hydantoinic  Parabanic  acid, 

acid. 

C8H2N208  +  2  H20  =  H2C204  +  CO(NH2)2. 


152  PHYSIOLOGICAL  CHEMISTRY. 

FORMULAE  OF  URIC  ACID  AND  ITS  CHIEF  DERIVATIVES. 


NH-C NH 

I 
CO 

NH— C 

I  I 

CO— NH 


C5N4N403. 


NH— CH— NH 

I 
CO 

I 
NH— CO    CO 

I 
NH2 


ALLANTOIN. 

(Glyoxyl-diureid). 


NH— CH 1 

i 

*H 

NH-CH— OH 

i 

1 
CO 

i 

CO 

1 

NH2    COOH  CO 

1 

NH— CO 

NH2 

ALLANTO 

IC   ACID. 

ALLANTURIC   ACID 

(Glyoxyl  Urea). 

C4H6N403. 


C4H8N404 


C3H4N2Os. 


NH— CO 

1                     1 

NH-CO 

NH— CO 

1          | 

NH-CO 

1 

1          1 
CO     CO 

1                  1 

CO     CO 

CO     CHOH 

1          | 

CO     CH2 

1          | 

1 

NH-CO 

NH2  COOH 

NH— CO 

NH— CO 

ALLOXAN. 

ALLOXANIC   ACID. 

DIALUEIC   ACID. 

BARBITURIC   ACID. 

(Mesoxalyl  Urea). 

(Tartronyl  Urea). 

(Malonyl  Urea). 

C4H2N204. 

C4H4N205. 

C4H4N204. 

C4H4N2Os. 

Alloxantin  (Alloxan  +  Dialuric  Acid),  C8H4N407. 

Purpuric  Acid  (Alloxan  +  Dialuramid),  C8H5N506. 

Ammonium  purpurate  or  murexid  (Alloxan  -f  Ammonium  dialuramid), 
C8H4N506  (NH4). 


NH— CO 

I 
CO 

I 

NH— CO 


NH— CO 

I 
CO 

NH2  coo: 


NH— CH2 
I 

co- 

I 

NH2  COOH 


NH— CO 

I     I 
CO  CH— NH2 

I    I 
NH— CO 


PARABANIC   ACID. 

(Oxalyl  Urea). 
C3H2N2Os. 


OXALURIC   ACID. 


C3H4N204. 


HYDANTOIC   ACID. 


C3H6N203. 


AMIDO-BABBITURIC  ACID. 

(Uramil,  Dialuramid). 


C4H5N3Os 


URINE.  153 

Xanthin  or  Nuclein' Bases. 

As  has  been  mentioned  under  uric  acid  (p.  139),  these 
bases  are  formed  from  nuclein  on  simple  cleavage  (see 
also  nucleo-histon,  and  alloxuric  bases).  They  have 
therefore  the  same  source  as  uric  acid.  Most  of  these 
bases  have  been  found  in  urine,  though  in  small  amount. 
Inasmuch  as  they  are  basic  substances  made  by  the  animal 
cell  they  belong  to  the  group  of  compounds  known  as 
leucomams.  The  following  table  will  serve  to  indicate  the 
close  relationship  that  exists  between  these  several  bases: 

Adenin,  C5H5N5.  Guanin,  C5H5N50. 

Hypoxanthin,  C5H4N4C\  Xanthin,  C5H4N402. 

(Uric  Acid,  C5H4N403). 

Heteroxanthin,  C6H6N402. 

(methyl  xanthin.) 

Paraxanthin,      C7H8N402. 

(di-methyl  xanthin 
or  uro-theobromin). 

It  is  interesting  to  note  that  theobromin,  the  active 
principle  of  theobroma  cacao,  is  also  a  di-methyl  xanthin 
and  is  isomeric  with  paraxanthin  of  the  urine.  Further- 
more, caffein  or  thein,  the  active  principle  of  coffee  and 
tea,  is  a  tri-methyl  xanthin  and  can  be  made  artificially 
from  xanthin — a  waste  product  of  the  animal  cell.  Adenin 
can  readily  be  converted  into  hypoxanthin;  and  guanin  can 
be  changed  by  the  same  process  into  xanthin.  The  conver- 
sion of  xanthin  into  uric  acid  has  long  baffled  the  chemist, 
but  this  has  at  last  been  accomplished  as  seen  by  the 
researches  of  Fischer.  All  the  members  of  this  group  may 
therefore  be  prepared  synthetically. 

.    .  NH CO 

Creatinin,  C4H7N30,    =    NH  =  C< 

N(CH3)  —  CH3. 

Creatinin  is  derived  from  creatin,  which  can  be  consid- 
ered as  a  cleavage  product  of    protein  matter.     Although 


154  PHYSIOLOGICAL  CHEMISTRY. 

creatin  is  abundant  in  muscles  (0.3  per  cent.)  it  is  not 
present  in  the  urine,  but  is  represented  by  its  anhydride 
creatinin.  This  dehydration  probably  takes  place  in  the 
kidney.  In  alkaline  urine  the  creatinin  may  be  changed 
back  into  creatin. 

The  chief  source  of  creatinin  in  the  urine  is  not  the 
creatin  in  the  muscles  of  the  body  but  rather  the  creatin 
present  in  the  food.  The  more  meat  eaten,  the  greater 
is  the  amount  of  creatinin  in  the  urine.  Milk  contains  no 
creatin  and  hence  when  this  is  used  as  the  only  food,  no 
creatinin  appears  in  the  urine.  The  mass  of  the  creatinin 
present  in  the  urine  cannot  therefore  be  considered  as  a 
waste  product  of  the  tissues. 

A  small  portion  of  the  creatinin  may  be  derived  from 
the  creatin  existing  in  the  muscles.  This  portion  can 
therefore  be  considered  as  a  waste  product  of  the  body. 
The  mass  of  the  creatin  existing  in  the  muscles  of  the 
living  body  undoubtedly  undergoes  disintegration  and  is 
eliminated,  probably  as  urea.  In  starvation,  however,  this 
is  not  true  since  creatinin  continues  to  be  excreted. 

An  adult  man  excretes  daily  about  1.0  g.  of  creatinin; 
women  excrete  about  one-third  less.  The  actual  amount  of 
nitrogen  that  thus  leaves  the  body  is  as  great  if  not  greater 
than  that  contained  in  uric  acid. 

Creatinin  is  increased  in  the  urine  in  diabetes,  due  to 
increased  meat  diet.  In  general,  the  excretion  of  creatinin 
is  parallel  with  that  of  urea  and  of  uric  acid. 

In  starvation  the  amount  of  creatinin  is  diminished 
somewhat  but  it  may  remain  close  to  the  normal.  As  the 
muscle  tissue  is  consumed  the  creatin  is  liberated  and 
passes  through  the  body  the  same  as  if  it  were  given  with 
meat  food.  For  this  same  reason  there  is  an  increase  in 
creatinin  in  the  urine  in  febrile  diseases.  In  convales- 
cence it  is  decreased.  After  ordinary  muscular  exercise 
creatinin  is  not  increased  in  the  urine;  but  if  the  exercise 
is  carried  to  exhaustion  an  increase  may  result.     In  such 


URINE.  155 

urine  a  leucomain,  xantho-creatinin,  C5H10N4O,  has  been 
found.  It  is  said  to  be  decreased  in  advanced  Bright's 
disease  and  this  retention  of  creatin  is  regarded  by  some  as 
one  of  the  causes  of  the  uraemic  symptoms. 

*The  following-  method  of  isolation  can  also  be  used 
for  quantitative  estimation  (Neubauer's  method):  200 — 300 
c.c.  of  urine  are  rendered  alkaline  with  calcium  hydrate 
and  then  calcium  chloride  is  added  to  completely  precipi- 
tate the  phosphates.  Baryta  mixture  can  be  used  instead 
of  calcium  chloride.  The  precipitate  is  filtered  off  and 
washed;  the  filtrate  and  wash- water  are  combined,  slightly 
acidulated  with  acetic  acid,  and  evaporated  to  a  syrup.  This 
while  warm  is  mixed  with  50  c.c.  of  95—97  per  cent,  alcohol 
and  the  mixture  transferred  to  a  beaker,  covered  and 
allowed  to  stand  eight  hours  in  the  cold.  The  precipitate 
is  then  filtered  off,  washed  with  alcohol,  and  the  filtrate 
and  washings  are  concentrated  to  50 — 60  c.c.  When  cold 
one-half  of  a  c.c.  of  a  zinc  chloride  solution,  of  a  specific 
gravity  of  1.20  and  free  from  acid,  is  added.  The  thoroughly 
stirred  mixture  is  covered  and  allowed  to  stand  in  a  cold 
place  for  two  or  three  days.  The  precipitate  is  collected 
upon  a  small  weighed  filter.  The  filtrate  can  be  used  to 
transfer  all  the  crystals.  These  are  then  washed  with  a 
little  alcohol,  till  all  the  chlorides  are  removed,  and  finally 
dried  at  100°  and  weighed.  100  parts  of  creatinin  zinc 
chloride  contains  62.42  parts  of  creatinin.  From  this  com- 
pound the  pure  creatinin  can  be  obtained  by  heating  with 
lead  hydrate.  The  solution  is  filtered,  decolored  with 
animal  charcoal,  evaporated  to  dryness  and  the  residue 
extracted  with  strong  alcohol  (creatin  remains  undissolved). 
The  alcohol  solution  can  be  concentrated  to  crystallization 
and,  if  need  be,  the  crystals  can  be  further  purified  by 
recrystallization  from  water. 


156  PHYSIOLOGICAL  CHEMISTRY. 

Hippuric  Acid,  C9H9N03,  =  C6H5CO.NH.CH2.C02H. 

Hippuric  acid  has  been  designated  as  a  facultative 
waste  product.  That  is  to  say,  it  is  not  a  direct  product 
of  tissue  change  but  is  made  in  the  body  whenever  benzoic 
acid  is  present.  Hippuric  acid  results  from  the  union  of 
benzoic  acid  and  glycocoll.  Glycocoll,  or  amido-acetic 
acid,  is  an  intermediate  nitrogenous  waste-product  which, 
as  an  amido  acid,  eventually  yields  urea  and  hence  does 
not  appear  in  the  urine.  If,  however,  some  substance  is 
present  which  will  unite  with  it  and  thus  protect  it  against 
oxidation  it  then  is  excreted  in  this  complex  form.  Ben- 
zoic acid  possesses  this  affinity  for  glycocoll  and  hence  if  it 
is  present  in  the  food,  or  is  formed  by  protein  decomposi- 
tion in  the  intestine,  or  is  directly  administered,  it  unites 
with  glycocoll  and  is  excreted  as  hippuric  acid.  A  similar 
conjugation  is  seen  in  the  glycocholic  acid  of  bile.  The 
glycocoll  in  this  case  unites  with  cholic  acid.  The  result- 
ant bile  acid  is  excreted  into  the  intestine  where  it  is  de- 
composed into  its  components.  The  glycocoll  thus  set  free 
may  be  reabsorbed  and  if  benzoic  acid  is  present  may  now 
yield  hippuric  acid.  The  source  of  the  glycocoll  present  in 
hippuric  acid  then  is  tissue  metabolism  and  it  is  obtained 
either  directly,  or  after  it  has  passed  out  with  the  bile  as 
glycocholic  acid. 

The  benzoic  acid  which  is  necessary  to  hippuric  acid 
formation  is  derived  first  from  benzoic  acid  and  its  deriva- 
tives that  may  exist  preformed  in  the  food.  These  com- 
pounds are  present  in  plant  foods,  especially  fruits  such 
as  plums,  prunes,  huckleberries,  mulberries,  cranberries, 
etc.  The  protein  molecule  contains,  as  is  indicated  by  the 
color  reactions,  an  aromatic  nucleus.  When  therefore 
the  protein  molecule  is  split  up  by  bacteria  various 
aromatic  bodies  result.  Phenyl-propionic  acid  is  thus 
formed  and  in  the  body  becomes  oxidized  to  benzoic  acid. 
Consequently  a  second  source  of  benzoic  acid  is  the  putre- 


URINE.  157 

faction  of  proteins  in  the  intestines.  Plant  food  is  not 
wholly  necessary  in  order  that  hippuric  acid  shall  form, 
since  with  an  exclusively  meat  diet,  or  in  starvation,  this 
acid  continues  to  be  elaborated.  It  is  possible  that  as  a 
third  source  very  small  amounts  of  benzoic  acid  are  formed 
by  tissue  metabolism.  The  fact  that  in  starvation  hippuric 
acid  is  still  eliminated  does  not  prove  that  benzoic  acid  is 
made  by  the  tissues,  since  even  then  bacterial  growth  in  the 
intestines  is  not  wholly  suppressed. 

The  interesting  studies  of  Nuttall  and  Thierfelder  on 
"sterile"  guinea-pigs  show  that  even  in  the  absence  of 
bacteria  from  the  intestines  aromatic  oxy-acids  are  elimi- 
nated with  the  urine  and  hence  are  in  part  waste  tissue 
products.  Other  aromatic  compounds  as  phenol,  indol, 
skatol,  pyrocatechin  and  probably  benzoic  acid  are  not 
present  and  are  therefore  to  be  considered  not  as  products 
of  tissue  metabolism  but  solely  of  intestinal  putrefaction. 

It  is  evident  that  glycocoll  is  an  intermediate  waste 
product  which  would  not  be  met  with  in  the  excretions 
were  it  not  for  the  presence  of  cholic  or  of  benzoic  acid. 
In  a  similar  manner  the  existence  of  other  intermediate 
waste  products  has  been  demonstrated.  As  already  men- 
tioned ammonia  is  one  of  these  and  does  not  appear  in  the 
urine  unless  mineral  acids  are  administered,  or  formed  by 
oxidation  of  proteins  in  the  body.  In  combination  as  a  salt 
of  a  mineral  acid  it  escapes  conversion  into  urea.  Car- 
bamic  acid  is  likewise  an  intermediate  waste  product  which 
normally  is  changed  into  urea.  If,  however,  calcium  hy- 
drate is  administered  calcium  carbamate  is  formed  and  is 
excreted.  The  existence  of  glycuronic  acid  as  an  interme- 
diate waste  product  was  established  in  a  similar  manner. 
When  camphor,  chloral,  naphthol  or  toluol  are  adminis- 
tered they  unite  with  glycuronic  acid  and  the  resultant 
compound  appears  in  the  urine. 

The  amount  of  glycocoll  present  in  the  tissues  is  not 
unlimited,  for  when  more  than  about  1  g.  of  benzoic  acid  is 


158  PHYSIOLOGICAL  CHEMISTRY. 

taken  some  of  this  acid  appears  in  the  urine  uncombined. 
Salicylic  acid  (hydroxy-benzoic  acid)  when  taken  internally 
appears  in  the  urine  as  such,  and  also  as  salicyluric  acid  (hy- 
droxy-hippuric  acid)  OH.C6H4CO.NH.CH2.C02H.  When 
benzoic  acid  is  given  to  birds  it  is  not  excreted  as  hippuric 
acid  but  as  ornithuric  acid,  C19H20N2O4.  The  latter  on  de- 
composition yields  benzoic  acid  and  ornithin,  CsH^NaOa- 
Ornithin  therefore  corresponds  to  glycocoll  and  is  probably 
a  di-amido-valerianic  acid. 

The  synthesis  of  benzoic  acid  and  glycocoll  to  hippuric 
acid  takes  place  in  the  kidney. 

Herbivorous  animals  excrete  much  more  hippuric  acid 
than  do  carnivorous  animals.  This  difference  is  in  part  due 
to  the  food  but  more  especially  to  the  marked  putrefactive 
changes  carried  on  in  the  intestines  of  herbivorous  animals. 
Their  intestines  are  large  and  pouchy,  or  sacculate,  as 
compared  with  the  smooth,  continuous  intestines  of  carniv- 
orous animals.  Consequently  the  remnants  of  food  will 
be  held  back  and  undergo  greater  putrefaction.  In  this  way 
is  explained  the  relatively  high  excretion  by  herbivorous 
animals  of  such  aromatic  putrefaction  products  as  phenol, 
indol,  skatol,  pyrocatechin,  hippuric  acid,  etc. 

Hippuric  acid  was  found  in  the  urine  of  man  for  the 
first  time  in  diabetes.  In  this  disease  it  is  present  in 
increased  amount,  due  undoubtedly  to  the  rich  protein  diet 
and  to  much  fruit.  In  fevers  it  is  decreased  and  probably 
also  in  kidney  diseases,  because  of  diminished  synthetic 
activity  of  the  kidney  cells.  It  is  not  decreased  in  icterus 
as  has  been  supposed. 

A  normal  individual,  with  mixed  diet,  excretes  daily 
about  0. 7  g.  of  hippuric  acid.  With  a  diet  rich  in  fruit  the 
amount  may  rise  to  2.0  g.  or  more.  When  present  in  the 
sediment  in  urine  it  forms  long  rhombic  prisms  or  needles, 
and  may  be  mistaken  for  uric  acid,  or  even  for  triple  phos- 
phates. By  attention  to  diet  and  to  intestinal  changes  the 
amount  can  be  promptly  diminished. 


URINE.  159 

Preparation  from  urine. — Boil  ]/* — 1  liter  of  urine  (of  the  horse 
or  cow)  with  excess  of  thin  milk  of  lime;  filter  while  hot  and  con- 
centrate the  filtrate  on  a  wire  gauze  to  ]4> — 1/%  the  original  volume. 
Cool,  add  excess  of  hydrochloric  acid  and  set  aside  for  24  hours. 
Filter  off  the  reddish  crystals  of  hippuric  acid  and  dry  them  between 
filter  paper.  Dissolve  the  crystals  of  crude  hippuric  acid  in  as 
little  water  as  possible  and  filter;  heat  the  filtrate  to  boiling  and 
pass  chlorine  gas  till  the  color  of  the  solution  becomes  pale-yellow. 
Then  cool  rapidly,  filter  and  wash  the  crystals  several  times  with 
cold  water.  Recrystallize  from  boiling  water  to  which  animal  char- 
coal has  been  added  (Curtius).  To  purify  the  crystals  boil  again 
with  milk  of  lime,  filter  and  reprecipitate  with  hydrochloric  acid. 
The  crystals  can  be  still  further  purified  by  recrystallization  and 
decoloring  with  animal  charcoal. 

Human  urine  can  be  employed  if  1 — L5  g.  of  benzoic  acid  have 
been  taken  the  previous  evening. 

Synthetic  preparation. — To  some  glycocoll  in  a  test-tube  add  a  little 
water,  then  a  few  drops  of  sodium  hydrate  and  about  1  c.c.  of  benzoyl 
chloride.  Close  the  tube  with  a  stopper  and  shake  vigorously  as  long 
as  the  tube  continues  to  become  hot.  Finally  render  strongly  alkaline 
with  sodium  hydrate  and  shake  until  the  odor  of  benzoyl  chloride  dis- 
appears. When  cold  acidulate  with  hydrochloric  acid,  add  an  equal 
volume  of  ether  (petroleum  ether  is  better)  and  shake  up  thoroughly 
in  order  to  extract  the  benzoic  acid.  Decant  the  ethereal  layer  into 
a  clean  porcelain  dish  and  repeat  the  extraction  as  before.  The 
aqueous  solution  with  the  insoluble  hippuric  acid  is  filtered,  and  the 
hippuric  acid  washed  slightly  on  the  filter  with  cold  water,  then  re- 
crystallized  from  a  small  amount  of  boiling  water  (just  sufficient  to 
dissolve). 

The  ethereal  solution  on  evaporation  yields  benzoic  acid. 

The  synthesis  takes  place  according  to  the  equation: 

CH,NH2  +  cocl.C6H5  =  CH,NH.CO.C6H5  +  Ha 
CO2H  C02H 

PROPERTIES  OP  HIPPURIC  ACID. 

Determine  the  melting-point.     What  is  it? 

Test  the  solubility  in  alcohol,  ether  and  water. 

Test  the  taste  and  reaction. 

Examine  the  crystalline  form  under  the  microscope. 


160  PHYSIOLOGICAL  CHEMISTRY. 

REACTIONS. 

*1. — Carefully  neutralize  a  solution  of  hippuric  acid 
with  sodium  hydrate,  and  add  a  drop  or  two  of  neutral 
ferric  chloride  solution  when  the  cream  colored  ferric  salt 
is  precipitated.  Test  the  solubility  in  hot  water,  hot 
alcohol  and  acids. 

2. — Boil  a  few  crystals  of  hippuric  acid  with  some  sodium  hypo- 
bromite  solution — a  kermes-brown  precipitate  forms  (distinction  from 
benzoic  acid). 

3. — Place  some  hippuric  acid  in  a  dish,  add  nitric  acid  and  evap- 
orate to  dryness.  Transfer  the  residue  tc  a  test-tube  and  heat  in  a 
Bunsen  flame.  Observe  the  odor  of  nitro-benzol  (artificial  oil  of  bitter 
almonds). 

4. — On  moderate  heating  in  a  test-tube  hippuric  acid  melts  to  an 
oily  fluid  which  on  cooling  solidifies  to  a  crystalline  mass.  On  stronger 
heating  the  liquid  becomes  red,  rises  along  the  walls  of  the  tube,  and 
yields  a  sublimate  of  benzoic  acid  and  an  odor  of  hydrocyanic  acid 
(peach-blossoms). 

5. — Place  1  g.  of  hippuric  acid  in  a  small  Erlenmeyer  flask,  add 
about  10  c.c.  of  dilute  sulphuric  acid  (1 — 2),  then  insert  a  perforated 
stopper  provided  with  a  condensing  tube  (Pig.  1,  p.  10.)  Heat  the 
contents  on  a  wire  gauze  at  just  below  the  boiling  point  for  some 
hours.  Observe  the  sublimation  of  crystals  of  benzoic  acid.  When 
the  decomposition  is  complete,  cool,  add  about  25  c.c.  water  and  filter 
off  the  benzoic  acid.  Extract  the  filtrate  with  ether  to  remove  the 
dissolved  benzoic  acid;  then  dilute  the  aqueous  liquid  with  water  and 
neutralize  on  the  water-bath  with  barium  carbonate.  Filter,  con- 
centrate the  filtrate  on  the  water-bath  almost  to  dryness,  and  set 
aside  to  allow  the  glycocoll  to  crystallize. 

Save  the  benzoic  acid  for  subsequent  tests. 

Complete  the  equation: 

C6H5.CO.NH.CH2.COaH  +  HaO  = 

Glycocoll. 

To  a  portion  of  the  glycocoll  dissolved  in  water  add  a  drop  of 
copper  chloride— a  blue  color  is  the  result.  What  effect  has  the  addi- 
tion of  sodium  hydrate  to  this  solution? 

To  another  portion  add  ferric  chloride — note  the  result. 


URINE.  161 

Benzoic  Acid,  C7H602,  =  C6H5.CO.OH. 

Benzoic  acid  may  be  present  with  hippuric  acid  in  the 
urine,  especially  when  the  glycocoll  necessary  to  make 
hippuric  acid  has  been  all  taken  up.  It  may  be  present, 
tog-ether  with  uncombined  glycocoll,  whenever  the  renal 
cells  are  unable  to  effect  the  synthesis,  as  is  the  case 
in  certain  diseases  of  the  kidney.  Furthermore,  in  alka- 
line urine  hippuric  acid  may  be  split  by  ferments  into  ben- 
zoic acid,  and  it  is  even  possible  that  a  ferment  (histozym) 
exerting  such  action  is  present  in  the  kidney  of  the  dog 
and  hog. 

The  sources  of  the  benzoic  acid  in  the  urine  have  been 
mentioned  under  hippuric  acid  (p.  156). 

Preparation. — This  acid  is  obtained  in  the  decomposition  of  hip- 
puric acid  (see  p.  160)  and  can  be  purified  by  recrystallization  from  hot 
water. 

1. — Examine  the  crystalline  form  as  it  is  obtained  (1)  from  hot 
water,  (2)  from  ether. 

2. — Heat  some  benzoic  acid  in  a  test-tube,  held  vertically  over 
the  flame — the  acid  sublimes  in  needles.     Note  the  odor  evolved. 

3.— Determine  the  melting-point  of  the  crystals. 

4.— Test  the  solubility  in  water,  alcohol,  and  in  ether. 

5. — To  a  solution  of  a  salt  of  benzoic  acid  add  neutral  ferric 
chloride  solution.  A  brownish  yellow  precipitate  forms — distinction 
from  salicylic  acid  which  gives  a  reddish  violet  color.  To  a  portion 
of  the  precipitate  add  ammonium  hydrate— it  dissolves  and  ferric 
hydrate  is  thrown  down  instead.  To  another  portion  add  hydrochloric 
acid  and  set  aside  over  night.     What  is  the  result? 

*6. — Place  some  benzoic  acid  in  an  evaporating  dish, 
add  nitric  acid  and  evaporate  over  a  flame  to  dryness;  then 
gently  heat — the  odor  of  nitrobenzol  (artificial  oil  of  bitter 
almonds)  will  be  perceived. 


162  PHYSIOLOGICAL  CHEMISTRY. 

7. — With  sodium  hypobromite  it  gives  no  precipitate — distinc- 
tion from  hippuric  acid. 

8. — Add  a  solution  of  benzoic  acid  or  of  its  salt  to  a  mixture  of 
alcohol,  barium  chloride  and  ammonia.  No  precipitate  forms— dis- 
tinction from  succinic  acid.  » 


Indoxyl,  C8H7NO,  =  C6H4  <^hH>CE 


This  occurs  in  the  urine  as  potassium  indoxyl  sulphate, 
C8H6N.O.S02.OK,  and  is  commonly  known  as  indican  or 
indigogen. 

Indoxyl  is  derived  from  indol,  which  is  formed  as  a  pro- 
duct of  the  putrefaction  of  protein  matter  in  the  intestine. 
Indol  when  absorbed  becomes  oxidized  to  indoxyl  which 
unites  with  sulphuric  acid  and  an  alkali  metal  and  is  then 
excreted  by  the  kidneys  as  an  ethereal  sulphate.  Indoxyl 
may  also  unite  with  glycuronic  acid  and  appear  thus  in  the 
urine. 

Indol,  skatol  and  phenol  are  ordinarily  decomposition 
products  due  to  bacterial  activity.  They  may  be  prepared 
by  fusing  proteins  with  potassium  hydrate.  The  several 
color  reactions  as  studied  in  connection  with  the  proteins 
(Exp.  6,  p.  40)  have  shown  the  existence  of  three  distinct 
aromatic  groups  or  nuclei  within  the  protein  molecule. 
These  three  are  the  phenol,  phenyl  and  indol  groups.  It 
is  evident  that  certain  proteins  when  acted  upon  by  bac- 
teria will  give  off  the  phenol  group  more  readily  than 
either  of  the  other  two;  or  the  reverse  may  occur.  The 
three  groups  of  decomposition  products  are  not  always, 
therefore,  in  the  same  relative  proportion  but  may  vary 
within  wide  limits  according  to  the  nature  of  the  bacteria 
at  work  and  the  material  being  acted  upon. 

Indoxyl  results,  as  has  been  stated,  by  the  oxidation  of 
indol.  On  further  oxidation  it  yields  indigo  which  may 
therefore   be   met   with   in   the   urine.      Indigo   on   reduc- 


URINE.  163 

tion  yields  indol.  The  amount  of  indoxyl  in  normal  urine, 
after  a  mixed  diet,  corresponds  to  from  5  to  20  mg.  of  indigo 
per  day.  It  is  much  more  abundant  in  the  urine  of  herbiv- 
orous animals,  as  the  horse.  This  is  true  not  only  of  indol, 
but  also  of  skatol,  phenol  and  conjugate  sulphates.  The 
reason  for  this  increase  of  putrefactive  products  is  given 
under  hippuric  acid.  An  exclusively  meat  diet  will,  of 
course,  be  followed  by  an  increased  excretion  of  these 
aromatic  compounds. 

Indoxyl  will  be  increased  materially  in  diseases  where 
the  peristaltic  action  of  the  intestine  is  diminished.  Like- 
wise in  obstructions  of  the  small  but  not  of  the  large  intes- 
tine. As  is  well  known  from  laboratory  experience  certain 
bacteria,  as  the  cholera  vibrio  and  the  bacillus  of  typhoid 
fever,  yield  an  indol  reaction.  Consequently,  in  certain  infec- 
tious diseases  of  the  intestines  the  amount  of  indol, and  hence 
of  indoxyl,  will  be  increased.  This  is  notably  the  case  in  the 
early  stage  of  cholera.  It  should  be  remembered  that  fre- 
quent diarrhoea  may  diminish  the  chance  of  absorption  of 
such  decomposition  products  and  hence  the  urine  may  con- 
tain less  aromatic  bodies  than  normal  urine.  Increased 
indoxyl  excretion  is  found  in  typhoid  fever,  in  intestinal 
tuberculosis,  in  carcinoma,  catarrh  and  dilatation  of  the 
stomach;  also  in  lead  colic,  in  pernicious  anaemia  and  in 
leukamia.  In  starvation  indoxyl  is  diminished  but  phenol 
may  not  only  persist  but  may  be  increased.  Bacterial  decom- 
position may  continue  in  the  intestines  during  starvation ;  the 
mucin  of  bile,  intestinal  secretions,  and  perhaps  intestinal 
haemorrhages  furnishing  material  for  the  bacteria  to  thrive 
upon.  This  material  may  not  give  off  the  indol  as  readily  as 
the  phenol  group,  and  hence  the  increase  in  phenol  excre- 
tion as  mentioned. 

Indoxyl  is  a  brownish  oil  which  is  not  permanent  but 
is  oxidized,  even  by  the  air,  to  indigo.  When  heated  in  the 
absence  of  air  it  yields  indoxyl-red.  In  alkaline  urine 
indoxyl  is  most  likely  to  undergo  conversion  .into  indigo. 


164  PHYSIOLOGICAL  CHEMISTRY. 

Indigo  may  occur  in  urine  in  solution,  in  the  sediment  or  in 
calculi. 

Jaffe's  Test  for  Indoxyl — To  10—20  c.c.  of  urine  in  a  test-tube  add 
an  equal  volume  of  concentrated  hydrochloric  acid,  a  few  c.c.  of 
chloroform,  then  drop  by  drop  a  dilute  solution  of  sodium  (or  cal- 
cium) hypochlorite.  Shake  after  the  addition  of  each  drop  and  avoid 
adding  an  excess.  The  chloroform  gradually  turns  blue  since  the 
indoxyl  has  been  oxidized  to  indigo-blue.  This  reaction  has  been  util- 
ized as  the  basis  of  a  quantitative  method. 

2  C8H7NO  +  2  O  =  C16H10N2O2  +  2  H2O. 

Apply  the  above  test  to  some  urine  from  the  horse  or  cow,  then 
to  human  urine. 

When  the  amount  of  indoxyl  or  indican  is  considerable 
it  can  be  estimated  by  weight.  That  is,  the  indigo  which 
separates  out  on  standing  is  washed  with  hot  water,  am- 
monia and  again  with  water,  then  dried  at  105° — 110°  and 
weighed.  It  can  also  be  estimated  colorimetrically,  or 
with  the  spectroscope. 

Indol  itself  forms  plates  which  melt  at  52°  and  are  easily 
soluble  in  hot  water,  alcohol,  and  in  ether.  It  possesses  a 
marked  fecal  odor.  The  solution  in  ligroin  gives  a  beauti- 
ful red  precipitate  with  picric  acid. 

TESTS  FOR  I^DOL. 

1. — The  solution  is  colored  red  by  nitric  acid  containing  a  trace 
of  nitrous  acid.     This  reaction  is  not  given  by  skatol. 

2. — A  pine  splinter  moistened  with  hydrochloric  acid  is  colored 
red  on  contact  with  an  alcoholic  solution  of  indol. 

3. — Millon's  reagent  gives  a  yellow  color,  whereas  with  skatol  it 
a  dirty-brown. 

£4. — Examine  the  laboratory  specimen  of  indol — note  the  odor. 


URINE.  165 

Skatoxyl,     C9H9NO. 

The  potassium  salt  of  skatoxyl-sulphuric  acid,  C9H8N. 
O.S02OK,  may  occur  in  the  urine.  Skatoxyl  is  not  known 
in  the  free  state. 

The  source  of  skatoxyl  is  skatol,  which  has  the  same 
origin  as  phenol,  indol,  etc. — namely  intestinal  putrefac- 
tion. The  skatol  on  absorption  is  oxidized  to  skatoxyl 
which  unites  with  sulphuric  acid  to  form  an  ethereal  sul- 
phate, as  in  the  case  of  phenol  and  indol.  Some  skatoxyl 
may  unite  with  glycuronic  acid. 

Urines  rich  in  skatoxyl,  on  standing-,  color  from  the 
surface  downward.  The  color  may  be  reddish,  violet  or 
black.  Such  urine  on  contact  with  HC1  gives  a  dark-red  to 
a  violet  color;  with  HN03  a  cherry-red  color.  Whereas 
the  indigo  formed  in  urine,  as  in  Jaffa's  test,  is  soluble  in 
chloroform  or  ether,  the  coloring  matter  derived  from 
skatoxyl,  under  the  same  conditions,  is  insoluble.  It  will 
be  dissolved  by  amyl  alcohol,  and  chloroform;  ether  will 
take  it  up  out  of  neutral  or  alkaline  solutions. 

Skatoxyl  and  indican,  in  urine,  may  yield  several  dis- 
tinct pigments  which  have  been  given  various  names,  such 
as  uroglaucin,  urocyanin,  urorubin,  urohamatin,  uroro- 
sein,  etc. 

DETECTION   OF   SKATOXYL. 

1. — To  the  suspected  urine  add  hydrochloric  acid;  if  skatoxyl  is 
present  a  dark  red  to  violet  color  will  appear. 

2. — Addition  of  nitric  acid  to  the  suspected  urine  will  produce  a 
cherry-red  color  if  skatoxyl  is  present. 

3. — Urine  containing"  much  skatoxyl  darkens  on  standing,  like 
"carbolic  acid  urine,"  and  later  becomes  reddish,  then  violet  to  black. 

Skatol,  or  methyl  indol,  is  a  putrefaction  product  of 
protein  matter.  It  may  be  formed  direct,  or  by  the 
reducing  action  of  bacteria  on  indol.  Inasmuch  as  this 
reducing  action  is  most  marked  in  the  large  intestine  it 


166  PHYSIOLOGICAL  CHEMISTRY. 

accounts  for  the  presence  of  skatol  in  abundance  in  that 
portion  of  the  intestines.  An  obstruction  in  the  small 
intestine  is  indicated  by  a  large  increase  in  indol,  whereas 
an  increase  in  skatol  points  to  the  large  intestine  as  the 
seat  of  the  trouble.  The  relation  of  indol,  skatol  and 
skatoxyl  can  be  seen  in  the  following  formulae: 

CH3  CH2.OH 

Cy3!4..  yCH  C6H4X  .CH  C6H4/  XCH 

XNHX  XNHX  XNH/ 

INDOL.  SKATOL.  SKATOXYL. 

Skatol  forms  plates  which  melt  at  95°.  It  is  difficultly 
soluble  in  water;  is  soluble  in  alcohol.  The  picrate  forms 
red  needles.  Skatol  is  soluble  in  HC1,  yielding  a  violet 
color.  On  heating  with  H2S04  it  yields  a  beautiful  purple- 
red  color.  It  possesses  an  intense  fecal  odor.  The  syn- 
thetic preparation,  however,  is  not  so  odorous.  On  boiling 
with  KOH  skatol  does  not  decompose,  whereas  indol  does. 
Both  indol  and  skatol  can  be  distilled  in  a  current  of  steam, 
and  are  precipitated  by  picric  acid  in  the  presence  of 
hydrochloric  acid. 

TESTS  FOR  SKATOL. 

1. — It  does  not  give  a  red  color  with  nitric  acid  which  contains 
nitrous  acid,  but  only  a  whitish  cloud. 

2. — It  does  not  color  a  pine  splinter  moistened  with  hydrochloric 
acid. 

3. — Note  the  odor  of  a  specimen  of  skatol. 

Phenol  and  Cresols. 

Phenol,  C6H5OH,  and  cresol,  C6H4.CH3.OH,  are  like 
indol,  skatol,  etc. ,  common  putrefaction  products  formed  in 
the  intestines.  When  absorbed  they  combine  with  sul- 
phuric acid  and  are  eliminated  as  ethereal  salts.     If  the 


UKINE.  167 

amount  of  sulphuric  acid  is  insufficient  they  may  unite 
with  glycuronic  acid. 

The  term  "phenol "  as  ordinarily  used  in  connection  with 
urine  includes  the  cresols.  The  latter  are  methyl  phenols 
and  are  present  in  larger  amount  than  is  ordinary  phenol. 
Of  the  three  possible  cresols  the  para-  compound  is  most 
abundant.  The  ortho-cresol  has  been  detected  in  urine. 
The  phenol  and  cresols  are  all  tested  for  together. 

The  source  of  "phenol"  may  be  preformed  aromatic 
compounds  in  the  food.  Vegetable  food  is  especially  rich 
in  such  compounds  and  hence  increases  the  amount  of  these 
bodies  present  in  the  urine.  Likewise,  the  administration 
of  various  coal-tar  products,  as  benzol,  salol,  creosote,  will 
increase  phenol,  etc.,  and  also  conjugate  sulphates.  Apart 
from  the  administration  of  aromatic  compounds  as  such,  or 
in  the  food,  the  only  remaining  source  is  the  bacterial  decom- 
position of  protein  matter  in  the  intestine.  There  is  very 
little  evidence  that  phenol  is  given  off  as  a  tissue  product. 
By  the  action  of  the  pancreatic  ferment,  or  of  bacteria, 
tyrosin  is  formed  out  of  the  protein  molecule  and  on 
oxidation  will  yield  phenol. 

The  amount  of  phenols  present  in  normal  urine  is  sub- 
ject to  great  variation  (17  to  51  mg.).  Increased  excretion 
of  phenol  is  met  with  in  various  intestinal  diseases  wherein 
bacterial  decomposition  of  the  contents  is  favored.  It  may 
be  absorbed  from  large  abscesses,  as  well  as  from  the 
intestines.  It  is  usually,  but  not  necessarily,  associated 
with  indican.  Thus,  in  starvation  the  amount  of  the  latter 
is  decreased,  whereas  phenol  may  be  increased.  As  pointed 
out  under  indol,  bacterial  activity  in  the  intestines  is  not 
suppressed  during  starvation. 

Phenol  and  indoxyl  are  apparently  diminished  in 
nephritis.  They  are  also  said  to  be  diminished  in  the 
urine  in  icterus,  although  conjugate  sulphates  (probably 
of  other  aromatic  compounds)  are  present.  It  is  interest- 
ing to  note  that  aromatic  substances  such  as  phenol,  indican, 

12 


168  PHYSIOLOGICAL  CHEMISTRY. 

etc. ,  are  not  increased  as  a  result  of  abnormal  fermenta- 
tions in  the  stomach. 

Urine  rich  in  phenols  takes  on  a  dark  brown  or  dark 
green  color— the  so-called  ' '  carbolic  urine. "  This  is  largely 
due  to  the  presence  of  products  like  hydroquinon. 

Potassium  Phenol  Sulphate,  C6H5.O.S02.OK. 

*Sijnthetic  Preparation. — Prepare  first  some  potassium 
pyrosulphate  as  follows:  To  10  g.  of  pulverized  potassium 
sulphate  in  an  evaporating-  dish  add  6  g.  of  concentrated 
sulphuric  acid  and  warm  gently  over  a  flame,  stirring  well, 
till  the  mass  dissolves;  then  gradually  raise  the  heat  till 
the  mass  remains  in  quiet  fusion.     Cool  and  pulverize. 

Now  place  in  a  flask  6  g.  of  potassium  hydrate  dis- 
solved in  8  to  9  c.c.  of  water  and  add  10  g.  of  phenol.  When 
all  is  dissolved,  cool  to  60 — 70°  and  add  gradually  and  in 
small  portions,  agitating  well,  12.5  g.  of  the  finely  pulver- 
ized potassium  pyrosulphate.  Keep  the  mixture  at  60 — 70°, 
with  frequent  shaking,  for  8  to  10  hours;  then  add  about 
50  c.c.  of  boiling  alcohol,  shake  thoroughly  and  filter  while 
hot.  The  filtrate  on  cooling  solidifies  to  a  mass  of  bright 
plates  of  potassium  phenol-sulphate.  Recrystallize  once 
or  twice  from  boiling  alcohol  and  finally  dry  the  crystals 
between  filter  paper  or  in  a  desiccator  over  sulphuric  acid. 

The  synthesis  of  this  salt  is  shown  by  the  equation: 

C6H5.OK  +  K2S207  =  C6H5.O.S02.OK  +  K2S04. 

*REACTIONS  OF  POTASSIUM  PHENOL  SULPHATE. 

1. — An  aqueous  solution  of  potassium  phenol-sulphate 
is  not  precipitated  by  barium  chloride  even  in  the  presence 
of  acetic  acid.  Why  not?  The  commercial  salt  will  give  a 
precipitate  owing  to  the  presence  of  sulphates. 

2. — To  an  aqueous  solution  of  the  salt  add  hydrochloric 
acid  and  heat  a  few  minutes,  then  add  barium  chloride. 


URINE.  169 

What  is  the  result?     Write  the  equation  to  represent  the 
change. 

3. — In  solutions  of  the  salt  test  for  phenol  with  bromine- 
water,  ferric  chloride,  and  Millon's  reagent.     What  is  the 

result? 

■4. — Distill  a  solution  of  the  same,  acidulated  with  hydro- 
chloric acid,  and  in  the  distillate  test  for  phenol. 

TESTS  FOR  PHENOL. 

1.— To  a  dilute  solution  of  phenol  add  a  drop  of  neutral  ferric 
chloride  solution — violet  color. 

2. — To  some  phenol  solution  add  bromine-water  to  a  permanent 
yellow  color— yellowish  white  precipitate  of  crystals  of  tri-brom- 
phenol,  C6H2Br3OH.     Examine  under  the  microscope. 

3.— To  the  phenol  solution  add  some  Millon's  reagent  and  warm 
till  the  precipitate  dissolves — beautiful  red  color.  Explain  the  beha- 
vior of  proteins  with  Millon's  reagent. 

Detection  of  Phenol  in  the  Urine. — To  about  250  c.c.  of  horse's  or 
cow's  urine  add  25  c.c.  of  sulphuric  acid  and  distil.  Examine  the  dis- 
tillate by  the  above  tests  for  phenol  (and  cresol),  and  for  aceton,  ap- 
plying- the  tests  given  on  page  178. 

Pyrocatechin,  C6H4(OH)2. 

This  is  ortho-dioxybenzol  (1:  2)  and  occurs  in  the  urine 
as  pyrocatechin  sulphate,  C6H402(KO.S02)2. 

It  is  present  usually  in  small  quantity  and  may  be 
entirely  absent.  It  is  more  abundant  in  horse  urine.  The 
protocatechuic  acid  present  in  plant  food  may  be  considered 
as  its  chief  source,  although  it  may  form  in  the  body  by  the 
incomplete  oxidation  of  phenol.  Consequently  the  admin- 
istration of  phenol,  benzol,  etc.,  increases  the  amount  of 
pyrocatechin  in  the  urine.  It  has  been  reported  in  transsu- 
dates, and  is  probably  present  in  the  supra-renals.  Ac- 
cording to  Halliburton  it  is  present  in  the  cerebro-spinal 


170  PHYSIOLOGICAL,    CHEMISTRY. 

fluid,  but  this  has  not  been  confirmed  by  Nawratski  who 
found  the  reducing-  substance  to  be  probably  glucose  (0.05 
per  cent.) 

It  has  been  frequently  observed  that  urine,  at  first  of 
normal  color  on  standing,  especially  if  alkaline,  turns  brown 
and  even  black.  This  change  begins  at  the  surface  and 
gradually  extends  downward.  This  condition  of  urine  has 
been  designated  as  alkaptonuria.  In  some  instances  at 
least  this  may  be  due  to  pyrocatechin;  in  others  to  uroleu- 
cinic,  or  more  often  perhaps  to  homogentisinic  acid.  Such 
urine  has  the  additional  peculiarity  that  it  reduces  Fehl- 
ing's  solution,  and  may  therefore  be  mistaken  for  diabetic 
urine. 

Pyrocatechin  crystallizes  from  water,  or  ether  in  prisms; 
from  benzol  in  broad  plates.  It  melts  at  104°  and  sublimes 
in  glistening  plates.  It  is  soluble  in  water,  alcohol,  and  in 
ether;  also  in  cold  benzol  (distinction  from  hydroquinon). 
It  is  precipitated  by  lead  acetate. 

With  a  solution  of  pyrocatechin  make  the  following 
tests: 

1. — To  afewc.c.  of  an  aqueous  solution  add  several  drops  of  potas- 
sium hydrate  solution;  set  aside  and  notice  the  change  that  takes 
place  in  the  course  of  an  hour.  What  is  this  change  due  to?  Add 
some  pyrocatechin  to  a  little  urine  and  repeat  the  test. 

2. — To  the  dilute  aqueous  solution  add  some  ferric  chloride.  The 
solution  becomes  dark  green,  then  black;  now  add  ammonium  hydrate 
to  alkaline  reaction.  What  is  the  result?  Acidify  with  acetic  acid 
and  the  original  color  returns. 

3. — To  about  1  c.c.  of  the  aqueous  solution  add  one  drop  of  furfurol- 
water,  then  add  slowly  1  c.c.  of  concentrated  sulphuric  acid  in  such 
a  way  that  it  runs  down  the  side  of  the  tube  and  collects  at  the  bot- 
tom.    The  liquid  becomes  cherry-red  in  color,  later  violet. 

4. — Boil  with  a  little  Fehling's  solution.  Observe  the  reduc- 
tion. Solutions  of  other  metals,  such  as  silver,  gold,  and  platinum 
are  likewise  reduced,  whereas  bismuth  is  not. 


URINE.  171 

*  Detection  of  di-oxy -benzols  in  urine. — The  urine  is 
acidulated  with  H2S04  and  concentrated  to  expel  phenol, 
then  filtered.  The  cool  filtrate  is  repeatedly  extracted  with 
ether.  The  ethereal  extracts  are  combined  and  distilled; 
the  residue  is  neutralized  with  BaC03  and  again  extracted 
with  ether.  The  ether  solution  is  evaporated,  the  residue 
dissolved  in  water  and  treated  with  lead  acetate,  avoiding 
excess.  The  precipitate  contains  pyrocatechin;  the  fil- 
trate, hydro-quinon.  The  washed  precipitate  is  suspend- 
ed in  water,  acidulated  with  H2S04  and  extracted  with 
ether.  This,  evaporated  and  recrystallized  from  benzol, 
gives  pyrocatechin.  The  filtrate  is  treated  in  similar 
manner  and  the  final  residue  recrystallized  from  hot 
benzol  gives  hydroquinon. 

Hydroquinon,  C6H4(OH)2. 

This  is  para-dioxybenzol  (1:4).  It  is  not  a  normal  con- 
stituent but  may  be  found  in  urine  after  administration  of 
benzol,  phenol,  or  hydroquinon  as  an  ethereal  sulphate. 
Such  urine  on  exposure  to  air  eventually  turns  dark — giving 
the  so-called  carbol-urine. 

Hydroquinon  crystallizes  in  rhombic  prisms  or  plates 
which  melt  at  169°.  The  solubility  is  much  the  same  as 
that  of  pyrocatechin.  It  is,  however,  soluble  in  hot  benzol 
and  is  not  precipitated  by  lead  acetate.  Like  pyrocatechin 
it  reduces  alkaline  solutions  of  metals.  It  does  not,  how- 
ever, reduce  bismuth  subnitrate. 

Test  a  little  hydroquinon  solution: 

1. — According  to  the  directions  given  above  under  (1)  and  (4). 

2. — Rapidly  heat  a  minute  portion  in  a  test-tube.  Observe  a 
violet  vapor  which  condenses  to  an  indigo-blue  sublimate. 

3. — Heat  some  of  the  solution  with  ferric  chloride.  It  is  oxidized 
and  the  penetrating  odor  of  quinon  is  observed. 


172  PHYSIOLOGICAL  CHEMISTRY. 

s s 

I  I 

Cystin,     (C3H6NS02)2,     =     CH3-C-NH2  NH2-C-CH3. 

C02H  C02H 

Cystin  is  an  organic  sulphur  compound  present  in  the 
urine  in  the  rare  condition  known  as  cystinuria.  It  is  not 
present  in  normal  urine,  though  a  cystin-like  substance  has 
been  found  in  very  small  amounts.  Normally,  the  sulphur 
of  the  protein  molecule,  after  passing  through  a  number  of 
intermediate  stages  or  compounds,  is  in  large  part  com- 
pletely oxidized  and  eliminated  as  sulphuric  acid.  In 
cystinuria  nearly  one-quarter  of  the  total  sulphur  may 
appear  in  the  urine  as  cystin.  It  may  therefore  be  consid- 
ered as  a  product  of  abnormal  cell  metabolism — an  inter- 
mediate waste  product  that  has  escaped  complete  oxidation. 
That  such  intermediate  compounds  exist  is  demonstrated 
by  feeding  brom-benzol  to  dogs,  in  which  case  a  substi- 
tuted cyste'in  appears  in  the  urine.  The  benzol  compound 
unites  with  cyste'in  and  protects  it  against  oxidation  in  the 
same  way  that  benzoic  acid  protects  glycocoll.  Cyste'in  is 
a-amido-thiolactic  acid. 

SH 

NH2— C— CH3 

I 
C02H 

On  oxidation  it  yields  cystin,  and  the  latter  in  turn  on 
reduction  with  nascent  hydrogen  gives  rise  to  cyste'in. 
When  cyste'in  is  fed  to  an  animal  it  is  oxidized  in  the  body 
and  the  sulphur  is  eliminated  in  part,  or  wholly,  as  a 
sulphate,  and  it  is  because  of  this  proneness  to  oxidation 
that  cyste'in  is  not  present  in  normal  urine. 

In  cystinuria  the  urine  and  feces  have  been  shown  to 
contain  one  or  both  of  the  well  known  ptoma'ins,  cadaverin 


URINE.  173 

and  putrescin.  These  bases  are  known  to  be  bacterial  pro- 
ducts and  formed  therefore  in  the  intestine.  They  are 
not  present  in  the  urine  or  in  the  feces  of  normal  persons 
nor  are  they  present  in  the  excreta  of  diseased  individuals 
except  in  cholera.  It  would  seem,  therefore,  as  if  a 
special  organism  were  present  in  the  intestines  in  cystin- 
uria,  and  that  its  products,  the  diamines,  have  to  do  with 
the  excretion  of  cystin.  The  latter  is  not  present  in  the 
feces.  It  is  a  product  of  cell  metabolism  within  the  body. 
It  is  probable  that  this  strange  association  of  diamines 
and  cystin  is  due  to  a  protecting  action  of  the  former, 
whereby  the  latter  escapes  oxidation.  The  diamines  and 
other  products  are  made  in  the  intestines  by  bacteria;  they 
are  absorbed  and  unite  with  cystin,  and  this  combination 
as  soon  as  it  is  excreted  by  the  kidneys  is  decomposed  into 
cystin  and  diamines. 

In  cystinuria  the  urine  is  usually  alkaline  or  very 
slightly  acid.  Cystin  when  present  in  urine,  owing  to  its 
difficult  solubility,  will  be  found  in  the  sediment  and  may 
even  give  rise  to  the  rare  cystin  stones.  No  other  patho- 
logical change  is  observed  in  cystinuria.  The  condition 
has  been  met  with  more  often  in  men  than  in  women,  and 
moreover  has  been  known  to  occur  in  several  members  of 
the  same  family.  Apart  from  its  presence  in  the  urine  in 
cystinuria,  cystin  has  been  found  once  in  beef  kidneys,  in 
the  liver  of  a  horse,  and  of  a  drunkard,  and  in  the  pancre- 
atic digestion  of  fibrin. 

Cystin  is  strongly  laevo-rotary.  On  reduction  with  tin 
and  hydrochloric  acid  it  yields  cystein,  which  may  be  pre- 
cipitated quantitatively  by  mercuric  chloride.  Cystin 
crystallizes  in  characteristic  colorless,  thin  six-sided  plates. 
In  addition  to  its  microscopical  appearance  cystin  can 
further  be  recognized  by  the  following  properties: 

1.— It  is  insoluble  in  water,  alcohol,  ether,  acetic  acid; 
soluble  in  mineral  acids  and  alkalis.   . 


174  PHYSIOLOGICAL  CHEMISTRY. 

2. — In  alkaline  concentrated  solution  with  benzoyl 
chloride  (see  p.  133)  it  gives  benzoyl  cystin.  This  method 
can  be  used  for  separating-  cystin  that  is  dissolved  in  the 
urine. 

3. — On  boiling-  with  potassium  hydrate  the  sulphide  of 
potassium  forms  and  may  be  recognized  by  bringing  it  in 
contact  with  a  bright  silver  coin,  or  by  adding  a  drop  of 
lead  acetate. 

4. — It  yields  no  murexid  test  (p.  145). 

Examination  of  cystin  calculi. — Pulverize  and  dissolve  in 
sodium  carbonate  solution  and  then  precipitate  this  with 
acetic  acid.  Collect  the  precipitate,  wash,  and  dissolve  in 
a  little  ammonia.  Set  this  solution  aside  in  a  watch-glass 
to  evaporate  spontaneously,  then  examine  microscopically. 


Diamines. 

Cadaverin,  C5HUN2  =  nh2.  ch2.  ch2.  ch2.  CH2.  ch2.  nh2. 
Putrescin,  C4H12N2  =  nh2.  ch2.  ch2.  ch2.  ch2.  nh2. 

These  two  diamines  were  discovered  in  1885  by  Brieger. 
They  are  basic  products  formed  in  the  bacterial  decom- 
position of  various  proteins  and  consequently  belong  to  the 
group  of  ptoma'ins.  They  are  usually  found  together,  and 
their  presence  in  the  urine  and  feces  in  cystinuria  indicates 
intestinal  origin.  They  have  been  found,  but  not  con- 
stantly, in  the  discharges  of  cholera  and  cholerine.  They 
are  not  present  in  normal  urine. 

These  bases  can  be  isolated  from  the  urine  of  cystin- 
uria by  means  of  the  benzoyl  chloride  reaction.  For  the 
method  and  other  details  see,  Vaughan  and  Novy,  Pto- 
ma'ins, etc.,  1896,  p.  325. 


URINE.  175 

Leucin  and  Tyrosin. 

These  amido  acids  are  not  present  in  normal  urine  but 
may  be  present  in  pathological  urines,  in  solution,  or,  if 
the  amount  is  considerable,  in  the  sediment.  Because^of  its 
greater  insolubility  tyrosin  is  more  likely  to  occur  in  the 
sediment.  The  urine  may  contain  these  compounds  in 
various  liver  diseases,  as  in  acute  yellow  atrophy  and  in 
phosphorus  poisoning;  also  in  typhus  fever,  small-pox,  and 
in  severe  anaemia.  For  isolation  and  identification  see 
pages  83  to  86. 

CO.  OH 
Oxalic  Acid,      C,H,04 ,     =       i 

CO.  OH. 

This  compound  occurs  in  the  urine  as  calcium  oxalate, 
and  although  present  in  small  amounts  it  is  nevertheless  a 
constant  constituent  of  normal  urine.  On  an  average 
about  20  mg.  are  excreted  per  da3r.  The  source  of  this 
oxalic  acid  is  not  clearly  understood.  When  oxalic  acid  or 
its  salts  are  administered  it  is  in  part  eliminated  as  such. 
Since  many  articles  of  food,  especially  vegetables,  as 
lettuce,  spinach,  asparagus,  tomatoes,  apples,  grapes,  etc., 
contain  this  acid,  it  is  commonly  held  that  the  oxalic  acid 
of  normal  urine  is  derived  from  that  contained  in  the  food. 
The  administration  of  remedies  like  rhubarb,  scilla,  senna, 
valerian,  etc.,  for  like  reason  increase  the  amount  of  oxalic 
acid.  On  the  other  hand  it  is  ascribed  to  an  incomplete 
oxidation  of  carbohydrates,  fats  and  proteins  and  even  of 
uric  acid. 

When  oxalic  acid  is  unduly  increased  in  amount  this 
condition  is  designated  as  oxaluria.  This  is  met  with  in 
diabetes  where  the  presence  of  oxalic  acid,  as  well  as  of 
sugar,  is  ascribed  to  a  lack  of  oxidation.  It  is  also  met 
with  in  icterus,  in  cases  where  there  is  diminished  meta- 
bolism, digestive   or    nervous   disturbances.      At   times  it 


176  PHYSIOLOGICAL    CHEMISTRY. 

may  be  present  in  urine,  in  excess,  without  any  other 
noticeable  symptom.  No  special  significance  is  attached  to 
small  amounts  of  oxalates.  The  chief  danger  lies  in  the 
formation  of  stones,  in  the  kidney  or  in  the  bladder. 

Oxalates  may  be  present  in  acid,  neutral  or  alkaline 
urine.  They  are  at  first  held  in  solution  by  the  acid  phos- 
phates. On  standing  and  cooling  these  are  changed  to 
neutral  phosphates  and  oxalates  are  deposited.  Calcium 
oxalate  usually  forms  small  bright  octahedra;  it  may  form 
dumb-bells,  discs,  or  may  be  amorphous.  Its  insolubility  in 
acetic  acid  distinguishes  it  from  phosphates. 

Preparation  of  calcium  oxalate. — To  about  200  c.c.  of  urine  add  a 
few  drops  of  saturated  oxalic  acid  solution,  then  set  aside  for  24 
hours.  Examine  the  sediment  under  the  microscope  for  the  charac- 
teristic octahedral  crystals,  and  for  other  forms. 

1. — Sketch  the  different  forms  of  calcium  oxalate. 

2. — Test  the  solubility  in  water,  acetic  acid,  hydrochloric  acid. 

Aceton,  CH3.CO.CH3. 

Aceton,  or  di-methyl  keton,  is  probably  a  constant 
constituent  of  urine.  The  amount  present  in  normal  urine 
is  about  10  mg.  per  day.  It  may  be  considerably  increased 
in  certain  diseases  and  this  condition  is  designated  as 
acetonuria.  An  increased  excretion  of  aceton  may  be 
expected  whenever  there  is  increased  protein  disintegration. 
Thus,  it  is  increased  in  starvation,  or  after  a  meat  diet.  A 
carbohydrate  diet,  because  of  its  saving  action  on  proteins, 
will  diminish  aceton.  It  may  be  increased  to  ten,  or  even 
forty  times  the  normal  amount,  in  diabetes;  it  is  also 
increased  in  febrile  diseases,  and  in  many  wasting 
diseases,  as  cachexia,  anaemia,  carcinoma,  etc.  It  is 
increased  by  chloroform  narcosis,  and  by  administration  of 
salacetol. 


URINE. 


177 


Aceton  is  also  met  with  as  a  product  of  fermentation 
of  carboh}rdrates  in  the  alimentary  tract.  Thus,  it  may  be 
found  in  lactic  acid  fermentations  in  the  stomach,  and  in 
the  intestines.  Hence  it  may  be  in  the  feces.  It  has  been 
found  in  febrile  blood  and  in  the  breath. 

The  chief  source  of  aceton  is  without  doubt  the  disin- 
tegrating protein  molecule.  One  of  the  early  products 
formed  is  ,3-oxy-butyric  acid,  which  may  be  oxidized  to 
acetacetic  acid,  and  this  in  turn  yields  aceton.  All  three  of 
these  substances  may  be  present  at  the  same  time,  as  in 


diabetes.     In  diabetes  2  to  5  or  even  10  g.  of  aceton  may 
be  excreted  per  day. 

Aceton  itself  is  a  liquid  which  boils  at  56 — 57°.  It  pos- 
sesses a  decided  fruit-like  odor  and  is  soluble  in  water, 
alcohol,  and  ether.  It  is  easily  oxidized  to  acetic  and 
formic  acids,  and  with  Ehrlich's  reagent  gives  a  red  color. 


Isolation  of  Aceton. — As  a  rule  aceton  cannot  be  tested 


178  PHYSIOLOGICAL  CHEMISTRY. 

for  directly  in  the  urine.  It  is  necessary  to  resort  to 
distillation.  For  this  purpose  250  c.c.  of  fresh  urine  (pre- 
ferably diabetic)  are  acidulated  with  dilute  acetic  acid  and 
.20-30  c.c.  of  liquid  are  distilled  off  (Fig.  3).  The  distillate 
is  then  subjected  to  the  following-  tests: 


1. — Lieben,s  iodoform  test. — To  the  aceton  solution  add  some 
iodine  in  potassium  iodide,  then  potassium  hydrate  till  the  iodine  just 
^clears  off— a  yellowish-white  cloud  results.  Allow  to  settle,  then 
•examine  for  iodoform  crystals — six-sided  plates  or  stellate  groups. 

To  a  few  c.c.  of  water  add  some  alcohol  and  test  for  iodoform  as 
just  given. 

Inasmuch  as  alcohol  may  be  present  in  urine  the  test  as  given  is 
not  positive.  The  following  modification  distinguishes  between  the 
two  compounds. 

2.— Gunning's  test.— Render  the  liquid  strongly  alkaline  with  am- 
monia, then  add  tincture  of  iodine,  drop  by  drop,  till  the  black  pre- 
cipitate of  nitrogen  iodide  forms.  On  standing  this  disappears  more 
or  less  rapidly  and  iodoform  crystals  remain  if  aceton  is  present. 

3. — LegaVs  nitropi°usside  test. — To  the  liquid  to  be  examined  add  a 
iew  drops  of  freshly  prepared  sodium  nitroprusside,  then  some  caustic 
alkali.  A  ruby-red  color  similar  to  that  given  by  creatinin  appears. 
On  acidulating  with  acetic  acid,  the  color  is  changed  to  wine-red  if 
aceton  is  present,  and  to  yellow  if  it  is  absent.  If  ammonia  is  used 
instead  of  caustic  alkali  aceton  will  give  the  color  reaction, 
whereas  creatinin  will  not. 

4. — PenzoldVs  indigo  test.— Dissolve  a  few  crystals  of  ortho-nitro- 
benzaldehyde  in  hot  water;  cool,  add  the  liquid  to  be  tested  and  then 
some  sodium  hydrate.  If  aceton  is  present  the  liquid  turns  yellow, 
then  green  and  finally  blue.  On  shaking  with  chloroform  the  blue 
color  is  taken  up. 

5.— To  some  mercuric  chloride  solution  add  alcoholic  potash.  A 
precipitate  of  the  oxide  forms.  Then  add  the  aceton  solution,  shake 
thoroughly  and  filter.  Test  the  filtrate  with  ammonium  sulphide  for 
mercury.     Aceton  dissolves  freshly  precipitated  mercuric  oxide. 


URINE.  179' 

Acetacetic  Acid,  C4H603,     =    CH3  .CO.CH2  .C02H. 

This  compound  is  probably  not  present  in  normal 'urine. 
It  is  not  as  common  in  febrile  urine  as  aceton.  It  may  be 
present  in  the  urine  in  chronic  diseases,  as  tuberculosis. 
Like  aceton  it  is  formed,  though  not  as  constantly,  by  the 
breaking-  down  of  protein  matter.  It  is  frequently  present 
in  diabetes  (cliaceturia). 

Acetacetic,  or  diacetic,  acid  is  a  colorless  liquid  soluble 
in  water,  alcohol  and  ether.  On  heating  with  water  it 
readily  decomposes  into  carbonic  acid  and  aceton.  Hence 
in  the  distillation  of  urine  the  aceton  that  is  found  may  in 
part  be  derived  from  the  breaking  down  of  this  compound. 

1.— To  10—50  c.c.  of  the  urine  (diabetic)  add  dilute  ferric  chloride 
as  long  as  a  precipitate  of  phosphates  forms,  filter  and  to  the  filtrate 
add  some  more  ferric  chloride— a  wine-red  color  indicates  acetacetic 
acid.     {Gttiu < nit's  test). 

2. — The  urine  on  distillation  yields  aceton. 

3. — ffiimer's  test.  To  the  urine  add  some  potassium  iodide  solu- 
tion, then  ferric  chloride  in  excess.  If  diacetic  acid  is  present,  on 
boiling  vapors  are  given  off  which  are  intensely  irritating  to  the  eyes 
and  nose. 

It  should  be  remembered  that  many  aromatic  com- 
pounds like  antipyrin,  salicylic  acid,  etc.,  give  a  similar 
reaction  with  iron.  The  reaction  if  due  to  diacetic  acid  is 
not  given  by  the  urine  if  this  is  allowed  to  stand  for  a  day 
or  two,  or  if  it  is  heated. 

^-Oxy-trutyric  acid,  C4H803. 

This  acid  is  absent  from  normal  urine.  It  may  be 
present  in  the  urine  of  acute  infectious  diseases,  and  in 
diabetes.  In  the  latter  disease  it  may  be  present  to  the 
amount  of  30—50  g.  per  day,  and  in  one  case  as  much  as 
226  g.  were  found.     It  is  combined  with  ammonia. 


180  PHYSIOLOGICAL  CHEMISTRY. 

Cholesterin. 

As  stated  on  p.  95  cholesterin  is  a  very  rare  con- 
stituent of  urine.  It  may  be  expected  when  fat  is  present 
in  the  urine,  as  in  chyluria.  For  its  recognition  and  isola- 
tion see  the  page  mentioned. 


Fats. 


Pats  may  be  present  in  the  urine  as  globules,  or  as 
needle-shaped  crystals.  The  nature  of  these  crystals  can 
be  readily  ascertained  by  gently  heating  a  specimen  on  a 
slide  when,  if  composed  of  fat,  they  melt,  forming 
globules.  The  solubility  of  the  globules  in  ether  will  at 
once  distinguish  these  from  leucin.  It  should  be  remem- 
bered that  fat  is  a  frequent  accidental  constituent  of  urine, 
as  when  urine  is  collected  in  an  unclean  vessel,  or  when 
passed  after  digital  examination,  or  after  the  use  of  a 
catheter.  Pathologically  fat  may  be  present  in  urine  as  a 
result  of  fatty  degeneration  of  the  kidney,  etc.  This 
condition  (lipuria)  is  met  with  in  many  acute  infectious 
diseases,  in  Bright's  disease,  in  phosphorus  poisoning,  etc. 
Epithelial  cells  and  casts  may  be  expected  at  the  same  time. 
Fat  may  be  present  in  the  urine  in  pregnancy. 

Fat  is  frequently  met  with  in  the  urine,  in  the  tropics 
and  sub-tropics,  in  the  condition  known  as  chyluria.  Such 
urine  may  be  milk-white  in  appearance,  or  may  be  colored 
red  by  blood  admixture.  Albumose,  pepton,  cholesterin, 
etc. ,  have  been  found  in  such  urine. 

Fatty  acids,  such  as  formic,  acetic,  butyric  are  appar- 
ently present  in  normal  urine  in  small  amount  (50  mg.  per 
day).  They  are  increased  after  a  carbohydrate  diet,  in 
fevers  (lipaciduria),  in  structural  diseases  of  the  liver,  as  in 
cancer,  or  syphilis.  They  are  considerably  increased  during 
ammoniacal  fermentation  of.  urine. 


URINE.  181 

Fats  ma3r  constitute  the  nucleus  of  urinary  calculi,  and 
in  rare  cases  such  calculi  may  consist  of  only  fat  and  fatty 
acids  (Horbaczewski). 

Carbohydrates. 

Urine,  normal  and  abnormal,  may  contain  a  number  of 
carbohydrates.  Thus,  glucose  is  a  constant  constituent  of 
normal  urine;  the  amount,  however,  rarely  exceeds  0.02 
per  cent,  and  is  therefore  not  recognizable  by  ordinary 
tests.  Iso-maltose  and  inosite  are  probably  present.  A 
dextrin-like  body  known  as  animal-gum  has  also  been 
found  in  normal  urine. 

Under  special  conditions  other  carbohydrates  may 
appear.  This  is  seen,  for  instance,  when  large  quantities 
of  glucose  (200  g.)  are  ingested.  In  a  few  hours  sugar  can 
be  detected  in  the  urine,  but  it  soon  disappears.  The  same 
is  true  when  laevulose,  or  cane  sugar,  or  lactose  are  taken 
in  large  amounts.  These  may  appear,  therefore,  as  such 
in  urine.  Lactose,  moreover,  may  be  present  in  the  urine 
in  pregnancy.  Fruit  rich  in  pectic  substances  (pentosanes) 
as  cherries,  plums,  etc.,  may  cause  the  appearance  of 
pentoses. 

An  increase  in  carbohydrates  in  general  can  be  detected 
in  the  urine  by  the  application  of  Molisch's  reaction  (Exp. 
1,  page  17);  also  by  the  benzoyl  chloride  reaction. 

Pentoses. 

These  sugars,  as  mentioned  above,  may  be  present  in 
the  urine  after  certain  fruit  diet.  They  have  been  found 
in  several  urines  (p.  16,  pentosuria):  in  diabetes,  and  as 
decomposition  products  of  a  nucleo-proteid  from  the  pan- 
creas. Such  urine  will  give  a  slow  reduction  on  prolonged 
heating  with  Fehling's  solution,  but  will  not  undergo  fer- 


182  PHYSIOLOGICAL   CHEMISTRY. 

mentation  if  glucose  is  absent.  The  separation  of  pentoses 
from  glucose  can  be  accomplished  by  preparing  the  osazons 
(p.  21).  On  digesting  the  mixed  osazons  with  water  at  60° 
the  pentosazon  dissolves  while  the  glucosazon  is  insoluble. 
The  former  can  then  be  recrystallized;  it  melts  at  157 — 160°. 
The  reaction  of  pentoses  with  anilin  acetate  is  given  on 
p.  16.  Another  reaction,  likewise  based  upon  the  forma- 
tion of  furfurol,  is  to  add  to  fuming  HC1,  saturated  with 
phlorogucin,  one-half  its  volume  of  urine.  The  mixture  is 
immersed  in  boiling  water.  The  foam  and  liquid  become 
colored  an  intense  red.  This  reaction,  it  should  be  remem- 
bered, is  also  given  by  glycuronic  acid. 

Glucose. 

As  already  stated  glucose  is  present  in  traces  in  normal 
urine.  When,  however,  it  is  increased  beyond  this  amount 
and  is  constant  in  the  urine  it  becomes  abnormal  (glycosuria). 
Sugar  may  thus  be  increased  under  various  pathological 
conditions,  as  in  lesions  of  the  brain  and  cord;  in  diseases 
of  the  heart,  liver  and  lungs;  in  cholera;  in  various  intoxi- 
cations as  from  morphine,  chloral,  carbon  monoxide,  etc. 

Persistent  excretion  of  sugar  is  met  with  in  diabetes 
mellitus.  The  amount  of  sugar  excreted  in  this  condition 
may  vary  from  a  trace  to  one-half  or  even  one  kilogram  in 
24  hour's  urine.  The  quantity  of  the  urine  is  greatly  in- 
creased, 3 — 5  litres,  or  more  per  day.  The  color  of  the  urine 
is  therefore  usually  very  pale.  The  absolute  amount  of 
urea  and  of  other  normal  constituents  is  likewise  greatly 
increased.  Consequently,  although  the  quantity  of  the 
urine  is  considerable,  the  presence  of  the  sugar  and  the 
increase  in  the  other  solids  impart  a  high  specific  gravity 
to  the  urine.  This  is  commonly  1.030 — 1.040  but  may  be 
much  higher. 

The  excretion  of  a  large  quantity  of  urine  which  pos- 
sesses a  very  light  color,  a  high  specific  gravity,  and  reduces 


URINE.  183 

Fehling's  solution  points  to  the  presence  of  sugar.  It 
should  be  remembered  that  normal  urine  may  give  a  slight 
reduction,  because  of  the  presence  of  reducing  sugars,  or  of 
uric  acid  and  creatinin,  or  of  glycuronic  acid  compounds. 
The  latter  may  appear  in  the  urine  in  increased  amount, 
as  conjugate  compounds,  after  administration  of  camphor, 
chloral  hydrate,  etc.  Furthermore,  in  alkaptonuria 
there  are  reducing  substances  as  uroleucinic  and  homo- 
gentisinic  acids.  Reducing  substances  also  appear  in  the 
urine  after  administration  of  rhubarb,  senna,  antipyrin, 
salol,  turpentine,  etc.  The  reaction  of  urine  with  copper 
or  bismuth  solutions  does  not  prove  that  sugar  is  present; 
it  merely  indicates  the  presence  of  a  reducing  substance 
which  may,  or  may  not,  be  sugar.  If  the  reaction  is  nega- 
tive one  can  correctly  conclude  that  sugar  is  absent.  The 
fermentation  test,  or  osazon  reaction  (p.  21),  can  be  em- 
ployed to  prove  that  the  reducing  substance  is  glucose. 

Fehling's  test  (Exp.  8  a,  p.  19)  is  commonly  employed 
for  the  detection  of  sugar.  Albumin  may  be  piesent  in 
diabetes.  Aceton,  acetacetic  acid,  oxy-butyric  and  fatty 
acids  are  also  present.  Diabetic  urine  on  standing  will 
frequently  contain  yeast-cells. 

Albumin  and  Globulin. 

Normal  urine  does  not  contain  these  or  other  proteins, 
except  mucin,  which  is  probably  a  nucleo-albumin.  Serum 
albumin  and  serum  globulin,  at  times  even  fibrinogen,  may 
appear  in  the  urine  under  a  variety  of  conditions.  These 
compounds  are  all  included  under  the  term  ''albumin." 
The  relative  amount  of  these  substances  is  not  constant; 
at  times  the  urine  may  contain  chiefly  albumin,  and  again 
globulin  may  predominate.  Apparently  albumin  predomi- 
nates when  diuresis  is  marked,  and  when  this  is  decreased 
globulin  is  increased.  The  quantity  of  albumin  and  globulin 
varies  greatly  from  the  merest  trace,  indicated  by  faint  cloud - 

13 


184  PHYSIOLOGICAL  CHEMISTRY. 

iness  when  acid  is  added,  to  such  quantities  as  will  cause  the 
urine  to  solidify  on  heating-.  Usually  the  amount  excreted 
in  a  day  is  a  fraction  of  a  gram,  but  may  be  increased  to  20 
or  even  30  grams.  It  is,  however,  usually  less  than  y2  per 
cent.  This  relatively  small  amount,  taken  into  considera- 
tion with  the  increased  urine  excretion  {polyuria)  explains 
why  the  specific  gravity  of  urine  in  albuminuria  is  low, 
1.010  or  even  less.  When  the  secretion  of  urine  is  dimin- 
ished, as  may  happen  in  the  later  stages  of  nephritis,  the 
specific  gravity  rises  and  may  reach  1.040  or  more. 

Temporary  albuminuria  may  occur  in  otherwise  healthy 
persons  as  a  result  of  improper  food,  nervous  disturbances 
and  excessive  muscular  exercise.  In  the  latter  case  the 
protein  present  is  probably  a  nucleo-albumin  since  it  is  pre- 
cipitated by  acetic  acid  in  the  cold.  The  presence  of  blood, 
pus,  and  semen  will  introduce  albumin  into  urine  and  such 
origin  is  detected  by  the  aid  of  the  microscope.  It  may 
also  occur  in  pregnancy. 

Albuminuria  proper  is  most  often  the  result  of  paren- 
chymatous degeneration  of  the  kidney,  and  is  accompanied 
by  casts.  It  occurs  in  acute  or  chronic  nephritis,  in  intoxi- 
cations from  substances  such  as  arsenic,  phosphorus,  strych- 
nine and  especially  bacterial  toxins.  Hence  febrile  albumin- 
uria is  a  common  condition  in  acute  infectious  diseases  as 
pneumonia,  erysipelas,  scarlet  fever,  small-pox,  yellow 
fever,  malaria,  diphtheria,  etc.  Usually  the  albuminuria 
disappears  with  the  fever.  In  such  cases  casts  are  present 
and  the  number  of  leucocytes  in  the  sediment  is  increased. 
Slight  albuminuria  has  been  met  with  in  ulcer  of  the  stomach ; 
in  icteric  urine,  where  it  is  accompanied  by  pale  yellow 
hyaline  casts  and  by  a  nucleo-albumin;  in  acute  yellow 
atrophy  of  the  liver;  in  anaemia  and  at  times  in  diabetes, 
carcinoma  and  in  rheumatism;  also  in  dyspnceic  condi- 
tions due  to  respiratory  and  circulatory  disturbances. 

Detection. — Before  applying  the  tests  for  albumin  the 


URINE.  185 

urine,  if  not  clear,  should  be  filtered.  Otherwise  a  slight 
cloudiness  resulting-  from  the  application  of  a  test  may- 
escape  unnoticed.  Should  the  urine  be  very  concentrated  it 
will  be  well  to  dilute  it  with  several  parts  of  water,  or 
better  with  dilute  salt  solution,  to  prevent  precipitation  of 
uric  acid,  or  of  urea  nitrate. 

The  best  test  for  albumin  is  the  nitric  acid  and  heat 
test  as  given  in  Exp.  9  a,  p.  43.  It  is  really  Heller's  test 
supplemented  by  heat  whereby  the  precipitate  or  cloudi- 
ness due  to  urates,  nucleo-albumin,  etc.,  is  dissolved.  After 
administration  of  balsam  of  tolu  or  copaiba  the  urine  may 
give  with  nitric  acid  an  opalescent  ring,  soluble,  however, 
in  alcohol.  It  should  be  remembered  that  the  presence  of 
salt  favors  the  precipitation  of  albumin. 

The  ferrocyanide  test  is  likewise  very  delicate.  Any 
mucin  or  nucleo-albumin  present  in  the  urine  should  first  be 
removed  by  acidulation  with  acetic  acid,  allowing  to  stand 
for  some  time,  then  filtering.  To  the  clear  filtrate  the 
reagent  can  then  be  added  (p.  43). 

The  separation  of  albumin  and  globulin  is  accomplished 
by  saturation  with  magnesium  sulphate  (p.  48).  25 — 50 
c.c.  or  more  of  the  urine  should  be  taken. 

Estimation. — For  the  quantitative  estimation  of  albumin 
and  globulin  see  Chapter  XI. 

Albumose. 

This  substance,  or  rather  group  of  hydrated  proteins, 
may  at  times  be  present  in  urine.  The  "pepton"  that  has 
been  met  with  in  urine  is  undoubtedly  an  albumose.  Such 
pepton,  or  rather  albumose,  may  be  given  off  by  disintegrat- 
ing leucocytes  as  in  large  abscesses;  it  becomes  absorbed 
and  is  then  promptly  excreted.  The  recognition  of 
"pepton  "  in  urine  in  case  of  a  deep-seated  abscess  is  impor- 


186  PHYSIOLOGICAL  CHEMISTRY. 

tant.  Albumose  frequently  appears  in  the  urine  during 
pregnancy  and  during  confinement. 

Albumoses  have  been  found,  though  not  constantly,  in 
febrile  diseases,  such  as  the  acute  infectious  diseases;  in  can- 
cer and  ulcer  of  the  stomach;  in  acute  yellow  atrophy  of 
the  liver  and  after  phosphorus  poisoning;  in  anaemia  and 
leukaemia;  and  in  acute  rheumatism. 

Typical  albumosuria  is  very  rare  and  thus  far  only 
about  six  cases  have  been  described.  In  nearly  all  of  these 
softening  of  the  bones  (osteomalacia)  existed.  The  crys- 
talline globulin  found  in  urine  in  the  sediment  and  described 
by  Bramwell  and  Patton  is  in  reality,  according  to  Huppert, 
hetero-albumose.  Albumose  had  been  met  with  once  before 
in  the  sediment  of  urine. 

Detection. — Only  fresh  urine  should  be  tested  for  albu- 
mose since  albuminous  urine  may  on  standing,  by  the 
action  of  ferments,  give  rise  to  albumose.  Urine  rich  in 
albumin  should  always  be  tested  for  albumose.  Albumose 
coagulates  at  about  60°  and  the  precipitate  redissolves  on 
boiling,  whereas  albumin  coagulates  at  about  70°  and  does 
not  redissolve  on  boiling. 

The  method  for  the  detection  of  albumose  is  given 
under  Exp.  3,  p.  50.  Also  compare  methods  given  under 
pepton. 

Pepton. 

What  has  been  termed  pepton  by  the  older  writers  is  in 
reality  albumose.  There  is  probably  no  positive  evidence 
that  true  pepton  has  been  met  with  in  fresh  urine.  Ac- 
cording to  Siegfried  anti-pepton  has  the  formula  C10H15N3O5 
and  is  identical  with  the  "  fleisch-saure  "  which  he  isolated 
from  meat  extracts. 


URINE.  187 

Histon. 

This  substance,  which  resembles  in  many  respects 
pepton,  was  first  obtained  by  Kossel  from  the  nuclei  of 
blood  corpuscles  of  birds.  Subsequently  it  was  studied  by 
Lilienf  eld  who  obtained  it  by  decomposing  the  nucleo-histon 
derived  from  lymphocytes.  With  ammonia  it  gives  an 
insoluble  precipitate  and  it  is  coagulated  by  boiling.  Histon 
has  been  reported  in  peritonitis;  in  the  later  stages  of 
pneumonia,  erysipelas,  scarlet  fever,  and  in  leukaemia. 

Detection. — The  urine  is  precipitated  with  alcohol;  the 
precipitate  is  washed  with  hot  alcohol  and  dissolved  in  boil- 
ing water.  This  solution  is  cooled,  acidulated  with  hydro- 
chloric acid  and  allowed  to  stand  for  several  hours,  then 
filtered.  To  the  filtrate  ammonia  is  added;  the  precipitate 
is  filtered  off  and  washed  with  ammonia  till  the  wash-water 
ceases  to  give  the  biuret  reaction.  The  precipitate  is 
dissolved  in  acetic  acid  and  tested  (1)  by  biuret;  (2)  by 
coagulation. 

Nucleo-albumin  and  Mucin. 

The  nucleo- albumins  are  soluble  in  water,  if  alkali  is 
present,  and  from  such  solution  they  are  precipitated  on 
saturation  with  magnesium  sulphate  (distinction  from 
nucleo-histon).  In  this  respect  they  resemble  globulin,  but 
they  contain  phosphorus  and  on  treatment  with  pepsin 
and  hydrochloric  acid  yield  nuclein.  They  are  thrown  out 
of  solution  by  acetic  acid  but  are  soluble  in  excess  of  the 
reagent  (distinction  from  mucin). 

Both  mucin  and  nucleo-albumin  are  probably  present  in 
minute  quantity  in  normal  urine.  The  mucin  frequently 
separates  from  the  urine  on  standing  as  a  very  delicate 
cloud.  Mucin  is  detected  by  adding  acetic  acid  to  the  urine 
previously  diluted  with  two  or  three  volumes  of  water.     If 


188  PHYSIOLOGICAL  CHEMISTRY. 

necessary  the  urine  should  first  be  filtered.  It  is  soluble  in 
excess  of  mineral  acid.  It  is  insoluble  in  excess  of  acetic 
acid  and  on  decomposition  gives  a  reducing"  substance  (see 
page  59) — distinction  from  nucleo-albumin  and  nucleo-his- 
ton.  Heller's  test  may  give  a  reaction  with  mucin  which 
might  be  mistaken  for  albumin.  Mucin  is  especially  pres- 
ent in  the  urine  of  women,  and  is  increased  in  affections 
of  the  mucous  membrane  of  the  urinary  passages. 

Nucleo-albumin,  often  called  mucin,  is  increased  in  the 
urine  of  febrile  diseases;  in  leukaemia,  catarrhal  icterus, 
and  after  severe  muscular  exercise. 

Fibrin. 

Fibrin  may  be  present  in  the  sediment  of  urine  as 
threads,  flakes  or  as  blood  casts.  It  may  be  recognized  by 
the  presence  of  blood  cells,  and  by  its  insolubility  in  cold 
dilute  acids  and  alkalis;  it  is  soluble  on  prolonged  heating 
(p.  113). 

Blood. 

As  a  result  of  haemorrhage  in  the  kidneys,  or  elsewhere 
along  the  urinary  tract,  blood  may  appear  in  the  urine. 
This  condition  is  designated  as  hcematuria.  The  amount  of 
blood  may  be  so  small  as  to  scarcely  alter  the  color  of  the 
urine.  On  the  other  hand  the  urine  may  be  bright  red,  in 
which  case  it  will  necessarily  be  cloudy.  It  should  be  remem- 
bered that  large  amounts  of  urates  may  give  a  reddish, 
cloudy  appearance.  Microscopic  examination  will  easily 
establish  the  presence  of  blood  cells  and  hence  of  haema- 
turia.  In  addition  to  the  corpuscles,  fibrin  floccules,  or  even 
blood  casts,  may  be  found.  Albumin  and  globulin  will,  of 
course,  be  present  in  such  urine. 

As  a  rule  the  microscope  is  all  that  is  necessary  to 
establish  the  presence  of  blood  in  the  urine.     The  guajac 


URINE.  189 

test  (p.  105)  and  Heller's  test  supplemented  by  the  hainin 
test  (p.  108)  may  be  used.  The  latter  will  be  given  also  in 
hemoglobinuria. 

The  place  o\  haemorrhage  may  be  Indicated  by  the  form 

of  the  blood  clot.  Thus,  if  narrow  blood  easts  are  present, 
they  point  to  the  kidney  as  the  seal  of  the  haemorrhage, 
especially  if  the  amount  of  blood  is  slight.  Large,  coarse 
blood  clots  clearly  are  not  formed  in  the  kidney.  The  pres- 
ence of  squamous  epithelial  cells  and  absence  ^\  casts 
point  to  the  bladder  or  urethra. 

Haemoglobin. 

At  times  the  urine  may  be  blood-red  in  color  and  yet 
not  due  to  actual  haemorrhage.  Normally  the  blood- 
pigment,  haemoglobin,  is  held  within  the  corpuscles,     if, 

however,  destruction  of  the  cells  takes  place,  as  in  exten- 
sive burns,  or  intoxication  with  arsine,  chlorates,  etc..  the 
haemoglobin  is  set  free  and  dissolves  in  the  plasma.  It  is 
then  a  foreign  substance,  as  much  so  as  if  it  were  injected 
into  the  blood-current,  and  is  consequently  excreted  by  the 
liver  and  by  the  kidneys.  The  presence  of  haemoglobin  in 
solution  in  the  urine  is  designated  as  hcemogloMnfiria.  Not 
infrequently  the  haemoglobin  may  be  converted  into 
methaemoglobin. 

This  condition  is  recognized  by  the  color  of  the  urine, 
the  absence  Of  blood  cells,  by  a  positive  reaction  with 
guajac  (p.  105),  and  by  the  spectroscope  (p,  102). 

Haematoporphyrin. 

This  pigment  may  be  spoken  of  as  hamiatin  deprived 
of  its  iron.  It  is  said  to  be  present  in  normal  urine  in  \ cry 
minute  quantity.  It  has  been  found  in  the  urine  in  rheuma- 
tism, Addison's  disease,  cirrhosis  of    the  Liver,  etc.;   and 


190  PHYSIOLOGICAL  CHEMISTRY. 

especially  is  it  present  in  the  urine  after  continued  admin- 
istration of  sulphonal  or  trional.  Such  urine  may  be  dark, 
or  brownish  red,  or  may  have  even  a  violet  tinge.  If  the 
amount  of  coloring  matter  is  small  the  urine  may  scarcely 
show  a  color. 

Detection. — To  25  c.c.  of  the  urine,  baryta  mixture  or 
sodium  hydrate  is  added  to  alkaline  reaction.  The  phos- 
phates are  precipitated  and  drag  down  the  haematopor- 
phyrin.  The  precipitate  is  washed,  then  digested  at  room 
temperature  with  alcohol  acidulated  with  hydrochloric  acid 
and  filtered.  The  filtrate  is  examined  before  the  spectro- 
scope for  the  characteristic  spectrum  of  acid  haematopor- 
phyrin  (see  Exp.  5,  p.  103). 

The  origin  of  this  compound  is  uncertain.  According 
to  Stokvis  blood  that  passes  into  the  intestines  is  there 
reduced  to  haematoporphyrin  which  is  then  absorbed  and 
excreted  by  the  kidneys. 

Methaemoglobin. 

This  modified  blood  pigment  is  formed  in  the  urine  from 
haemoglobin.  It  is  present  quite  often  in  haemoglobinuria. 
It  is  recognized  by  the  spectroscope,  but  care  must  be 
taken  not  to  confound  it  with  haematin,  (see  Exp.  4,  p.  103). 
The  change  in  the  spectrum  after  addition  of  ammonium 
sulphide  distinguishes  it  from  haematin. 


Pus. 


Normal  urine  may  frequently  contain  occasional  leuco- 
cytes. As  a  result,  however,  of  various  inflammatory 
conditions  in  the  kidneys,  bladder  or  urethra  pus  may 
appear  in  variable  quantity  (pyuria).  If  formed  in  the 
kidney  or  bladder  it  becomes  distributed  throughout  the 


URINE.  191 

urine  and  consequently  the  last  portion  of  urine  that  is 
passed  will  be  as  rich  in  pus  corpuscles  as  the  first  portion. 
In  urethral  affections,  however,  the  first  portion  of  the 
urine  will  contain  nearly  all  the  pus  cells  and  the  last 
portion  will  be  almost  free  of  such  cells. 

The  presence  of  pus  in  urine  is  often  indicated  by  an 
abundant,  slimy  sediment.  Such  urine  always  contains 
albumin. 

1. — Microscopic  Examination. — This  is  the  quickest  and  best 
way  for  the  detection  of  pus.  The  pus  cells  are  larger  than  blood 
corpuscles:  they  are  colorless,  and  round  or  crenated.  One  or  more 
nuclei  can  be  seen,  especially  if  acetic  acid  is  applied.  With  iodine 
in  potassium  iodide  the  nuclei  turn  a  mahogany  brown.  In  alkaline 
urine  the  contents  of  the  cell  (nucleo-histon)  are  largely  dissolved  out 
and  hence  the  cell  itself  and  the  nuclei  may  be  scarcely  recognizable. 
Such  urine  on  the  addition  of  acetic  acid  will  give  a  precipitate  of 
nucleo-histon.  A  slimy  sediment,  together  with  albumin,  is  there- 
fore indicative  of  pus. 

2. — Donne's  test. — To  the  sediment  obtained  on  standing,  or  by 
centrifugation,  add  a  small  piece  of  potash  and  stir.  A  very  slimy, 
sticky  mass  results. 

3. —  VitaWs  guajac  test. — Cover  the  sediment,  acidulated  if  need 
be,  with  a  layer  of  guajac  tincture.  A  blue  coloration  develops.  Or, 
filter  through  a  small  filter,  then  pass  through  the  filter  a  few  drops 
of  the  guajac  tincture.     The  paper  and  filtrate  turn  blue. 


Nucleo-histon. 

This  substance  has  been  shown  by  Lilienfeld  to  be  the 
chief  constituent  of  the  nuclei  of  the  cells  of  the  thymus 
gland  and  other  tissues.  On  treatment  with  acids  and  other 
agents  it  readily  splits  up  into  nuclein  and  histon.  The 
complex  character  of  nucleo-histon  can  best  be  seen  from 
the  following-  scheme,  which  shows  at  the  same  time  the 
successive  products  of  decomposition: 


192  PHYSIOLOGICAL    CHEMISTRY. 


'  Histon  (protamin  and  proteid?). 
(basic) 


Nucleo-histon  < 


Proteid. 


LNS!,in       Nucleinic 
i      etc  id* 


f  Adenin. 
Nuclein     \ 

bases  t.  Guanin,etc. 

f  Thymin. 
L  Thymic     J  Cytosin. 

acid  1  Lasvulinic  acid. 
[  Phosphoric  acid. 


Nucleo-histon  has  been  isolated  from  the  urine  in  z*  case 
of  pseudo-leukaemia  (Jolles).  It  may  be  always  expected 
in  alkaline  urine  containing  pus.  When  present  in  urine 
it  is  precipitated  on  the  addition  of  acetic  acid  and  can 
therefore  be  mistaken  for  nucleo-albumin.  If  the  precipitate, 
however,  is  dissolved  in  dilute  sodium  carbonate  and  the 
solution  saturated  with  magnesium  sulphate  the  nucleo- 
albumin  is  precipitated,  whereas  the  nucleo-histon  remains 
in  solution.  That  the  precipitate  is  not  mucin  can  be 
demonstrated  by  the  absence  of  reducing  power  after 
decomposition  (as  in  Exp.  13  jb,  p.  59). 

To  isolate  nucleo-histon,  a  large  volume  of  urine  is 
heated  at  60—70°  for  %  hour,  then  filtered.  The  nitrate  is 
cautiously  acidulated  with  acetic  acid  and  then  thoroughly 
shaken  to  cause  the  flocculent  precipitate  to  settle.  The 
precipitate  is  filtered  off,  dissolved  in  dilute  sodium  hydrate 
(4  per  cent.)  and  filtered.  The  filtrate  is  precipitated  with 
acetic  acid  as  before,  and  this  purification  is  repeated  a 
third  time.  The  precipitate  is  finally  shaken  with  an  equal 
volume  of  absolute  alcohol,  filtered,  washed  with  warm 
alcohol,  then  with  ether  and  finally  dried. 

To  identify  the  product,  treat  it  for  some  hours  with  1 
per  cent,  hydrochloric  acid  in  the  cold  and  then  filter.  His- 
ton will  be  present  in  the  filtrate  and  can  be  recognized  (1) 
by  giving  a  precipitate  on  the  addition  of  ammonia;  (2)  by 
coagulating  on  heating,  the  precipitate  being  soluble  in 
acids;  (3)  by  giving  the  biuret  reaction  in  the  cold. 


URINE.  193 

Bile. 

The  bile  acids  and  bile  pigments,  which  constitute  the 
characteristic  constituents  of  bile,  are  elaborated  in  the 
liver  and  under  normal  conditions  they  are  passed  with  the 
bile  into  the  intestines.  As  a  result  of  intoxication  with 
phosphorus,  arsenic,  bacterial  toxins,  etc.,  or  of  closure  of 
the  bile  duct  by  catarrhal  exudates,  bile  stones,  etc.,  the 
constituents  of  the  bile  pass  into  the  blood.  The  pigments 
in  this  case  induce  the  condition  of  jaundice,  or  icterus. 
The  bile  constituents  in  the  blood  are  in  part  taken  up  by 
the  kidneys  and  eliminated  with  the  urine. 

The  bile  acids  probably  do  not  exist,  even  in  traces,  in 
normal  urine.  They  may  be  present  in  icteric  urine,  though 
this  is  not  always  the  case.  The  small  amount  of  these 
acids  when  present,  and  the  organic  constituents  of  the 
urine  render  it  difficult  to  detect  these  acids  in  urine.  The 
method  for  their  detection  in  urine  is  given  under  Exp. 
6  c,  p.  91. 

The  bile  pigments  impart  more  or  less  color  to  the 
urine.  It  may  be  yellowish  green  to  brown,  and  on  shaking 
a  yellowish  or  greenish  foam  results.  Such  urine  may  be 
cloudy  and  may  contain  albumin;  colored  casts  or  epithelial 
cells  and  granules  may  be  in  the  sediment.  Bilirubin  has 
been  identified  in  such  urine.  Other  bile  pigments  which 
are  easily  derived  from  bilirubin  may  also  be  present.  The 
tests  given  under  Exp.  7,  p.  92,  are  delicate  and  can  be 
employed  with  satisfactory  results. 

Ehrlich's  Diazo-reaction  for  Typhoid  Urine. 

The  reagent  employed  for  this  reaction  should  be  freshly  pre- 
pared.    The  following-  two  solutions  are  first  made. 

1.— To  1,000  c.c.  of  water  add  50  c.c.  of  concentrated  HC1  and 
1  g.  of  sulphanilic  acid. 


194  PHYSIOLOGICAL   CHEMISTRY. 

2. — A  0.5  per  cent,  solution  of  sodium  nitrite.  The  nitrite  solu- 
tion is  subject  to  oxidation  on  standing,  and  should  not  therefore  be 
prepared  in  large  quantity. 

Just  before  use,  to  form  the  reagent  proper,  these  two  solutions 
are  mixed  as  follows:  To  250  c.c.  of  solution  No.  1  add  5  c.c.  of  solu- 
tion No.  2.  Or,  on  a  smaller  scale,  to  5  c.c.  of  No.  1  add  3  or  4  drops 
of  solution  No.  2. 

Mix  the  urine  with  an  equal  volume  of  the  reagent,  and  add  at 
at  once  an  excess  of  NH4OH.  A  pink  to  a  deep  red  color,  and 
especially  a  pink  colored  foam,  constitutes  the  diazo-reaction. 

Normal  urine,  as  a  rule,  gives  a  brownish  yellow,  very 
rarely  a  pinkish  color.  The  reaction  is  very  rare  in 
chronic  non-febrile  diseases.  It  is  met  with,  as  a  rule, 
except  in  very  light  cases,  in  typhoid  fever,  and  a  cer- 
tain diagnostic  value  is  therefore  ascribed  to  this  reaction. 
It  has  been  found,  however,  in  exanthemic  typhus,  in  small- 
pox, in  acute  miliary  tubeculosis,  in  severe  tuberculosis, 
and  in  pneumonia.  The  disappearance  of  the  reaction  in 
typhoid  urine  may  be  taken  as  a  favorable  sign,  while  the 
appearance  of  the  reaction  in  tuberculosis  is  an  unfavora- 
ble indication. 

The  substance  which  gives  this  reaction  is  unknown. 
It  is  an  aromatic  compound,  probably  a  metabolic  product 
which  appears  in  the  urine  only  under  certain  special 
conditions. 

The  reaction  resembles  somewhat  the  test  for  nitrites 
as  given  in  Exp.  11,  p.  58.  If  naphthylamin  is  replaced  by 
a-naphthol  the  reaction  is  even  more  similar. 

Sulphur. 

The  proteins  of  the  food  and  of  the  tissues  constitute 
almost  the  sole  source  of  the  sulphur-containing  waste 
products.  A  small  amount  of  waste  sulphur  compounds  is. 
eliminated   as   sulphocyanate  by  the  saliva,  gastric  juice,. 


URINE.  195 

etc.  Another  small  portion  leaves  the  body  as  taurin  in 
the  taurocholic  acid  of  bile.  With  these  exceptions  almost 
all  the  sulphur  resulting  from  protein  disintegration 
appears  in  the  urine.  For  the  sake  of  convenience  the 
sulphur  compounds  of  the  urine  are  divided  into  two  groups: 
(1)  oxidized  or  acid  sulphur;  (2)  unoxidized  or  neutral  sul- 
phur. 

The  first  group,  or  oxidized  sulphur,  contains  com- 
pounds in  which  the  sulphur  is  present  as  sulphuric  acid, 
S03.  This  is  the  chief  form  in  which  sulphur  is  excreted. 
Thus,  while  the  total  sulphur  in  a  day's  urine  may  be  as 
high  as  1.0  g.,  the  sulphur  present  as  S03  averages  0.8  g. 
(2  g.  S03).  This  group  is  in  turn  sub-divided  into  (a)  simple 
sulphates,  such  as  potassium,  sodium,  magnesium,  calcium 
sulphates;  (&)  conjugate,  or  ethereal  sulphates.  In  the 
latter  the  sulphuric  acid  is  in  combination  with  an  organic 
radical  such  as  phenol,  indol,  skatol,  etc.  The  former  is 
precipitated  by  BaCl2,  the  latter  is  not.  Calcium  sulphate 
ma}''  appear  in  the  sediment  as  bundles  of  long  needles  not 
unlike  those  of  tyrosin.  The  relative  amounts  of  these 
two  groups  is  expressed  by  an  average  ratio  of  10  to  1. 
Consequently  the  24  hours'  urine  contains  0.1 — 0.2  g.  of 
S03  as  conjugate  sulphate. 

The  source  of  the  sulphur  in  the  sulphates,  as  indicated 
above,  is  the  sulphur  contained  in  the  proteins  of  the 
food  and  of  the  tissues.  Inasmuch  as  the  sulphates  con- 
tain most  of  the  waste  sulphur  it  follows  that  the  total 
sulphates  in  the  urine  furnish  an  excellent  index  of  pro- 
tein disintegration.  On  the  other  hand  the  organic  radicals, 
such  as  phenol,  indol,  etc.,  present  as  conjugate  sulphates 
owe  their  origin  either  to  the  administration  of  aromatic 
bodies  such  as  creosote,  carbolic  acid,  etc. ;  or  to  the  intes- 
tinal putrefaction  of  proteins.  The  protein  of  the  food 
may  undergo  bacterial  decomposition  in  the  intestines,  and 
phenol,  indol  and  other  aromatic  compounds  may  thus  be 


196  PHYSIOLOGICAL  CHEMISTRY. 

formed.  These  aromatic  compounds  are  probably  not 
formed  in  the  breaking  down  of  the  tissue  proteins.  Con- 
sequently the  amount  of  sulphur  present  as  conjugate 
sulphates  indicates  the  extent  of  intestinal  putrefaction. 
Conjugate  sulphates  are  present  in  the  urine  of  herbivorous 
animals  in  large  amount.  They  are  increased  in  the  urine 
of  man  by  intestinal  obstruction,  as  in  constipation,  and 
are  decreased  after  cathartics  and  in  starvation. 

The  second  group,  unoxidized  or  neutral  sulphur  com- 
pounds, includes  all  the  sulphur  bodies  except  sulphuric 
acid.  Some  of  these  compounds,  as  thiosulphates  and 
sulphocyanates  are  easily  oxidizable,  whereas  others  like 
taurin,  are  difficultly  oxidizable.  As  included  under  the 
head  of  neutral  sulphur  may  be  mentioned,  also  hydrogen 
sulphide,  cystin,  cystin-like  bodies,  sulphonic  acids,  ethyl 
sulphide,  mucin,  etc.  The  neutral  sulphur  in  the  urine  of 
man  averages  about  15  per  cent,  of  the  total  sulphur.  In 
the  urine  of  the  dog  it  is  more  than  double  this  amount. 

Hydrogen  Sulphide. 

It  is  possible  for  this  compound  to  appear  in  the  urine, 
eliminated  directly  from  the  blood.  In  such  a  case,  severe 
intoxication  necessarily  exists  and  death  results.  A  second 
source  of  hydrogen  sulphide  is  the  decomposition  that  may 
exist  in  neighboring  pus  cavities;  diffusion  takes  place 
through  the  walls  of  the  bladder  and  the  gas  is  absorbed 
by  the  urine.  In  both  instances  the  urine  is  likely  to  be 
perfectly  clear.  On  the  other  hand,  if  hydrogen  sulphide 
is  present  and  the  urine  is  cloudy  it  indicates  a  fermenta- 
tive decomposition  of  the  urine  analogous  to  the  ammon- 
iacal  fermentation.  In  this  case  bacteria  have  been 
introduced  into  the  bladder  through  a  rectal  fistula,  or  by 
a  catheter.  They  decompose  certain  of  the  neutral  sulphur 
compounds  and  yield  hydrogen  sulphide.  Several  micrococci 


URINE.  197 

and  bacilli  have  been  isolated  from  such  hydrogen  sulphide 
urine  (hydrothionuria).  This  property  belongs  not  merely 
to  a  few  organisms,  but  rather  to  most  bacteria.  Thus,  a 
large  number  of  the  common  non-pathogenic  and  patho- 
genic germs  will  give  rise  to  this  gas  when  grown  in  urine. 
Hydrogen  sulphide  can  be  detected  by  its  odor  and  by 
its  blackening  a  bright  silver  coin.  A  much  more  delicate 
test  is  to  suspend  in  the  neck  of  the  bottle,  from  a  slit  in  a 
cork  or  from  a  cotton  plug,  a  strip  of  filter  paper  previ- 
ously dipped  in  an  alkaline  lead  acetate  solution.  Black- 
ening promptly  results  if  the  gas  is  present. 

Sulphuric  Acid,     H2S04. 

To  about  10  c.c.  of  urine  in  a  test-tube  add  some  acetic  acid, 
then  some  barium  chloride  solution — barium  sulphate  is  precipitated- 
Now  filter  and  to  the  filtrate  add  hydrochloric  acid  (1  c.c);  boil  for  a 
few  minutes,  and  set  aside.  Another  precipitate  forms.  What  is  it? 
Explain  the  reaction. 

The  experiment  shows  that  sulphuric  acid  exists  in  the  urine  in 
at  least  two  forms — as  ordinary  sulphate,  and  as  ethereal  sulphate. 

Write  the  equations: 

BaCl2  +  K3SO,  = 

C6H5.O.S02.OK  +  H20  =  KHS04  + 

Prepare  and  examine  under  the  microscope  crystals  of  calcium 
sulphate,  made  by  adding-  a  few  drops  of  calcium  chloride  to  some 
urine  and  allowing  it  to  stand  for  a  short  time. 

*  "Neutral"  sulphur  can  be  tested  for  as  follows: 
Filter  off  the  barium  sulphate  precipitate  which  forms 
in  the  hydrochloric  acid  solution  as  described  above  and 
from  the  filtrate  remove  the  barium  by  careful  addition  of 
sodium  carbonate  solution.  Filter,  evaporate  the  filtrate 
to  dryness,  and  fuse  the  residue  with  potassium  nitrate  and 
hydrate.  Dissolve  the  fused  mass  when  cold  in  water, 
acidulate  with  hydrochloric  acid  and  precipitate  with  bar- 


198  PHYSIOLOGICAL,  CHEMISTRY. 

ium  chloride;  the  neutral  sulphur  has  been  oxidized  to  sul- 
phuric acid.  If  the  quantity  of  urine  taken  be  known,  and 
the  weight  of  the  precipitate  be  determined,  the  amount  of 
neutral  sulphur  can  be  estimated. 

Hyposulphurous  (Thiosulphuric)  Acid,     H2S203. 

Make  the  following  tests  with  a  solution  of  sodium  hyposul- 
phite: 

1. — Warm  some  of  the  solution  with  hydrochloric  acid — notice 
the  odor,  and  the  cloudiness  due  to  separation  of  free  sulphur. 
Complete  the  equation: 

Na2S203  +  2  HC1  = 

This  test  may  be  applied  to  urine  suspected  of  containing-  hypo- 
sulphurous  acid,  as  that  of  a  cat,  or  dog".  The  milky  appearance  on 
standing  is  due  to  free  sulphur. 

2. — To  a  few  c.c.  of  the  solution  add  a  few  drops  of  silver  nitrate 
solution.  The  solution  on  standing  becomes  black  from  reduced 
silver. 

3. — Add  1  or  2  drops  of  neutral  ferric  chloride  to  some  of  the  solu- 
tion.    What  is  the  result? 

4. — To  some  hyposulphite  solution  add  barium  chloride,  then 
alcohol — the  difficulty  soluble  barium  salt  forms. 

Phosphoric  Acid,     H3P04. 

This  acid  exists  in  the  urine  in  combination  with  so- 
dium, ammonium,  potassium  (alkaline  phosphates)  and  with 
calcium  and  magnesium  (earthy  phosphates).  A  small 
amount  of  phosphoric  acid  may  exist  in  ethereal  combina- 
tion as  glycerin-phosphoric  acid,  or  as  lecithin. 

Phosphoric  acid  (H3P04)  forms  three  series  of  salts: 
Normal— M3P04. 
Mono-hydric— M2HP04. 
Di-hydric— MH2P04. 


URINE.  199 

The  latter  salt  is  acid  in  reaction,  and  as  NaH2P04  it  is 
present  in  urine  and  to  it  the  acidity  of  this  secretion  is 
largely  due.  Sixty  per  cent,  of  the  total  phosphorus  in 
urine  exists  as  a  di-hydric  salt. 

The  sources  of  phosphoric  acid  are:  first,  the  preformed 
phosphates  in  the  food;  second,  the  phosphorus  in  organic 
combination  as  proteins,  nucleins,  lecithin,  etc.  Excess  of 
earthy  bases  in  the  food  may  result  in  precipitation  of  the 
phosphates  in  the  intestine,  in  which  case  they  are  excreted 
with  the  feces,  and  hence  the  urine  will  be  poor  in  phos- 
phates.    This  is  true  in  the  case  of  herbivorous  animals. 

The  quantity  of  phosphoric  acid  in  the  urine  is  there- 
fore subject  to  considerable  variation.  On  an  average  2.5 
g.  of  P2Os  are  excreted  per  day.  About  two-thirds  of  this 
amount  is  combined  with  alkali  metals,  the  remainder  with 
earthy  bases.  The  earthy  phosphates  are  held  in  solution 
by  the  acid  phosphates  and  by  salt;  when  the  reaction 
becomes  neutral  or  alkaline  they  are  precipitated.  Excess- 
ive muscular  exercise  is  attended  with  increase  of  phos- 
phoric acid. 

The  phosphoric  acid  is  diminished  in  amount  in  febrile 
affections  such  as  the  acute  infectious  diseases;  also  in 
diseases  of  the  kidneys,  because  of  non-elimination.  It  is 
increased  in  meningitis  and  in  diabetes. 

Neutral  Calcium  Phospnate,  Ca3(P04)2. 


1. — Heat  some  urine  in  a  test-tube.  Observe  that  it  becomes 
cloudy;  add  a  drop  of  nitric  acid  and  the  cloudiness  dissolves.  What 
is  it  due  to? 

2. — To  some  urine  add  sodium  hydrate;  a  precipitate  of  the 
phosphates  of  calcium  and  magnesium  is  thrown  down.  Examine 
the  precipitate  under  the  microscope.  What  is  its  appearance? 
Test  the  solubility  of  the  precipitate  in  acetic,  hydrochloric  and 
nitric  acids.  How  would  you  distinguish  between  a  deposit  of  amor- 
phous phosphates,  amorphous  urates,  and  amorphous  oxalates? 

14 


200  PHYSIOLOGICAL  CHEMISTRY. 

*  Acid  Phosphate  of  Calcium,  CaHPO^ — To  a  solution  of 
calcium  chloride  add  some  di-sodium  hydric  phosphate 
solution,  drop  by  drop.  Examine  carefully  the  character- 
istic form  of  the  crystals.  This  salt  is  always  crystalline 
and  is  deposited  in  slightly  acid  urine  only. 

To  some  acid  urine  add  dilute  ammonium  hydrate  till 
only  a  faint  acid  reaction  remains.  Set  aside  till  crystals 
form,  then  examine  under  the  microscope. 

Magnesium  Ammonium  Phosphate,  MgNH4P04. 

TRIPLE  PHOSPHATE. 

1. — To  some  urine  add  ammonium  hydrate  and  set  aside  over 
night.  Examine  under  the  microscope  for  stellate  or  pennate  crys- 
tals of  triple  phosphate. 

2. — Set  some  urine  aside  for  a  few  days.  Ammoniacal  fermenta- 
tion sets  in  and  the  prismatic  form  of  triple  phosphate  is  deposited. 
Examine  the  characteristic  crystals. 

This  salt  is  not  deposited  in  the  urine  unless  ammonia  is  present. 
The  reaction  may  be  neutral  or  alkaline.  If  stellate  crystals  of 
triple  phosphate  are  found  in  a  urine  what  does  it  indicate?  Are 
they  of  importance?  When  prismatic  crystals  of  triple  phosphate 
are  found  what  does  it  show?    When  are  they  of  importance? 

How  would  you  distinguish  the  short  prismatic  form  of  triple 
phosphate  from  crystals  of  oxalate  of  lime? 

Chlorides. 

Sodium  chloride  is  the  chief  form  in  which  hydrochloric 
acid  exists  in  the  urine.  The  other  bases  combine  with  but 
relatively  small  amounts  of  this  acid.  It  is  probable  that 
a  variable  amount  of  chlorine  (10 — 40  per  cent.)  exists  in 
an  organic  combination. 

The  amount  of  chlorides  in  the  urine  depends  primarily 
upon  the  quantity  of  these  salts  contained  in  the  food. 
Furthermore,  increased  exercise,  and  ingestion  of  water 
are   followed  by  increased   excretion   of    chlorides.      The 


URINE.  201 

amount  of  sodium  chloride  excreted  by  an  adult  in  24  hours 
will  necessarily  vary  greatly.     It  is  usually  10 — 15  g. 

Chlorides  may  be  markedly  decreased  in  acute  febrile 
conditions;  likewise  in  diarrhoea,  or  when  large  exudates 
or  transudates  form.  On  the  other  hand  chlorides  are 
increased  after  crises,  or  when  exudates  are  being  absorbed. 
A  decrease  in  chlorides  is  met  with,  further,  when  there  is 
lack  of  absorption  on  the  part  of  the  stomach  or  intestines; 
or  when  there  is  insufficient  excretion  by  the  kidneys. 

The  chlorides,  because  of  their  solubility,  are  never 
met  with  in  the  sediment  in  urine. 


Urinary  Sediment. 

Normal  acid  urine,  at  the  time  of  passage,  is  usually  a 
perfectly  clear  solution,  free  from  visible  suspended  or 
insoluble  matter.  If  the  urine  is  neutral  or  alkaline  in  re- 
action it  will  usually  be  cloudy  owing  to  the  precipitation 
of  phosphates.  A  cloudy  acid  urine,  due  to  suspended  bac- 
teria, is  met  with  in  acid  fermentation,  as  in  hydrothionuria. 
The  normal  acid  urine,  on  cooling,  may  undergo  a  change  in 
reaction,  due  to  the  fact  that  owing  to  mass  action  di-hydric 
phosphates  take  the  sodium  away  from  the  urates  and  thus 
form  mono-hydric  phosphates  which  impart  a  less  acid 
or  even  neutral  reaction  to  the  urine.  As  a  result  of  the 
withdrawal  of  sodium  free  uric  acid  is  deposited.  It  should 
be  remembered  that  about  0.7  g.  of  uric  acid  may  be  held 
in  solution  in  the  urine  by  the  urea  and  the  acid  phos- 
phates. From  normal  urine,  when  allowed  to  stand  for 
some  time,  there  invariably  separates  a  light  cloud  of 
mucin  which  may  contain  a  few  mucous  corpuscles.  Urine 
may  therefore  be  perfectly  clear  when  passed  and  may  give 
rise  to  a  sediment  on  subsequent  cooling  and  standing;  or, 
the  urine  may  be  cloudy  from  suspended  matter  at  the  time 
of  passage. 


202 


PHYSIOLOGICAL    CHEMISTRY. 


The  suspended  matter  in  urine  may  consist  of  a  great 
variety  of  substances,  many  of  which  possess  great  diag- 
nostic importance.  In  order  to  make  a  microscopic  exam- 
ination it  is  necessary  to  allow  the 
urine  to  stand  for  some  hours,  as 
over  night,  in  a  conical  test-glass. 
The  suspended  matter  then  settles  to 
the  bottom  and  can  be  removed  with 
a  pipette.  Much  time  can  be  gained 
and  better  results  obtained  by  the 
used  of  some  one  of  the  numerous 
forms  of  hand  centrifuges  (Fig.  4). 
The  suspended  matter  can  thus  be 
sedimented  in  2 — 3  minutes.  In  mak- 
ing microscopic  examinations  one 
should  be  on  guard  and  be  able  to 
recognize  the  presence  of  foreign 
material,  such  as  fatty  globules  or 
crystals;  starch  granules;  cotton, 
linen,  wool  or  silk  threads;  hair  and 
the  like. 

It  is  convenient  to  divide  the  substances  that  may  be 
present  in  the  sediment  into  two  groups, — organized  and 
unorganized. 


Organized  Sediment. 


Under  this  head  are  included  those  substances  which 
are  made  up  of  cells;  that  is  to  say  they  are  organized. 
Casts  are  described  under  this  head  although  they  are  not, 
strictly  speaking,  organized. 

Epithelial  cells  may  be  expected  in  small  numbers  in 
every  urine.  The  large,  fiat  squamous  cells  are  especially 
met  with  in  the  urine  of  woman.  Epithelial  cells  from 
different  parts  of  the  urinary  tract  may  be  present  in  large 


URINE.  203 

numbers.  Their  form  and  size  may  often  indicate  their 
origin.  Thus,  the  squamous  and  cylindrical  forms  may  be 
derived  from  the  urethra,  vagina,  or  bladder.  Numerous 
small,  roundish  cells,  frequently  grouped  and  present  in 
albuminous  urine,  are  derived  from  the  uriniferous  tubules. 
In  alkaline  urine  the  cells  may  undergo  partial  solution  and 
are  therefore  less  distinct.  They  can  be  stained  with 
dilute  anilin  dyes. 

Mucous  corpuscles  and  wandering  leucocytes  can  be  expect- 
ed in  small  numbers  in  normal  urine.  With  iodine  solution 
the  nuclei  take  on  a  mahogany  brown  color  (glycogen 
reaction). 

Pus  corpuscles,  or  altered  leucocytes,  when  present  in 
appreciable  numbers,  indicate  an  acute  or  chronic  inflam- 
matory condition  of  some  part  of  the  urinary  tract.  The 
cells  are  round  and  usually  have  2  or  3  nuclei  which  are 
rendered  distinct  by  the  application  of  acetic  acid.  In 
alkaline  urine  the  contents  dissolve  more  or  less,  and  render 
the  liquid  slimy.  This  is  often  the  case  in  cystitis.  Albu- 
min always  accompanies  pus  corpuscles  in  urine.  The  pus 
may  be  derived  from  the  urethra,  from  the  bladder,  or  from 
the  kidneys.     For  further  tests  see  p.  191. 

Blood  corpuscles  will  be  present  in  the  urine  whenever 
there  is  a  haemorrhage  in  some  portion  of  the  tract.  Albu- 
min and  globulin  are  therefore  likewise  present.  This 
condition,  haematuria,  is  to  be  distinguished  from  haemo- 
globinuria.  An  abundant  deposit  of  urates  may  impart  a 
reddish  color  to  the  sediment.  The  cause  of  haemorrhage 
is  to  be  ascertained  if  possible.  In  some  cases,  as  in 
tropical  haematuria,  the  cause  may  be  an  animal  parasite; 
in  others  the  tubercle  bacillus,  or  other  organism  may  be 
the  causal  factor.  The  place  of  haemorrhage  may  often  be 
indicated  by  the  form  of  the  clots,  or  blood  casts,  as  they 
are   called.      Long,  slender   blood   casts   accompanied   by 


204  PHYSIOLOGICAL  CHEMISTRY. 

renal  epithelial  cells  would  indicate  that  the  trouble  is  in 
the  kidney. 

Tissue  debris,  or  irregular  masses  of  cells,  are  often  met 
with  in  cancer  of  the  bladder;  less  often  in  cancer  of  the  kid- 
ney; likewise  in  tubercular  affections  of  these  organs,  or 
when  animal  parasites  are  present.  In  such  cases  blood  is 
frequently  present,  or  the  blood  pigment  may  be  altered 
and  appear  as  the  yellow  rhombic  plates  of  haemato'idin. 
Furthermore*  bacterial  invasion  may  exist,  hence  pus  cor- 
puscles and  putrid  decomposition  are  often  present.  The 
recognition  of  a  "cancer  cell"  in  the  urinary  sediment  is 
not  possible. 

Spermatozoa  may  be  present  in  urine  in  spermatorrhoea. 
They  possess  active  motion  and  can  easily  be  recognized  by 
the  microscope.  They  can  be  stained  by  the  ordinary  ani- 
lin  dyes.  When  present  in  urine  this  may  be  more  or  less 
cloudy  and  will  show  mucous-like  threads.  The  recogni- 
tion of  spermatozoa  is  of  considerable  medico-legal  impor- 
tance in  suspected  criminal  coition. 

Casts. — As  a  result  of  inflammatory  changes  the  epithe- 
lium lining  the  uriniferous  tubules  gives  off  or  allows  the 
passage  of  exudative  products  which  solidify  in  the  tubule 
and  thus  form  a  cast.  Eventually  the  pressure  of  liquid 
forces  the  cast  out  of  the  tube  and  it  is  then  carried  away 
with  the  urine.  Casts  are  usually  found  in  albuminous 
urine.  It  should  be  remembered,  however,  that  casts  may 
exist  in  urine  without  albumin  being  present.  This  is  not 
infrequently  the  case  after  prolonged  exercise,  such  as 
bicycling.  It  is  customary  to  distinguish  between  hyaline, 
granular,  epithelial  and  ivaxy  or  amyloid  casts. 

Hyaline  casts  are  colorless,  transparent,  homogeneous. 
They  are  usually  narrow,  but  may  be  wide  and  have  a  wavy 
border.  Owing  to  their  transparent,  colorless  appearance 
they  may  be   easily  overlooked.      The  diaphragm  should 


URINE.  205 

therefore  be  constricted  so  as  to  shut  off  most  of  the  light. 
They  may  be  rendered  more  visible  by  the  application  of  a 
dilute  anilin  dye.  With  iodine  they  color  yellow.  They 
are  soluble  in  acetic  acid  and  in  this  respect  are  different 
from  mucin  threads.  When  present  in  icterus  they  may 
have  a  yellowish  color.  They  may  be  present  in  severe 
fevers,  accompanied  by  albumin.  They  may  also  be  pres- 
ent in  urine  when  no  albumin  or  epithelial  cells  can  be 
detected. 

Granular  casts  are  rather  variable  in  size  and  color. 
They  may  be  considered  as  hyaline  casts  in  which  granular 
detritus  has  become  imbedded.  They  are  grayish  to  yel- 
lowish in  color. 

Epithelial  casts  are  merely  one  or  the  other  of  the 
above  forms  which  are  covered  in  part  or  entirely  by  epi- 
thelial cells.  The  inflammatory  process  has  loosened  the 
epithelial  cells,  which  adhere  to  the  cast  more  firmly  than 
to  the  basement  membrane  and  hence  are  eventually  ex- 
pelled adherent  to  the  cast. 

Waxy  or  amyloid  casts  are  comparatively  rare.  They 
are  more  refractive  than  the  hyaline  casts  and  have  a  dull 
yellowish,  homogeneous  appearance.  They  are  usually 
rather  broad  and  have  a  wavy,  twisted  contour.  They  con- 
sist of  amyloid  and  are  more  resistant  to  acids  than  the 
preceding.  Treated  with  iodine  they  take  on  a  reddish 
brown  color,  and  if  previously  exposed  to  sulphuric  acid 
they  turn  violet  (amyloid  reaction).  They  are  met  with  in 
several  forms  of  nephritis,  and  in  contracted  and  amyloid 
kidneys. 

In  addition  to  the  types  of  casts  as  just  described  vari- 
ous modifications,  or  mixed  casts,  are  met  with.  These 
may  be  briefly  alluded  to. 


206  PHYSIOLOGICAL  CHEMISTRY. 

Blood  casts,  or  clots  formed  within  the  tubules,  are 
met  with  in  acute  nephritis.  The  blood  corpuscles  are  held 
in  the  fibrin  meshes  or  are  cemented  by  albuminous  matter. 
The  cast  may  then  be  part  hyaline  and  part  blood.  Long- 
blood  casts'  are  not  infrequently  mistaken  for  worms. 

Pus  or  leucocyte  casts  may  likewise  result  from  admix- 
ture of  these  cells  with  a  hyaline  matrix. 

Fatty  casts  are  usually  hyaline  cylinders  containing- 
fatty  globules  or  bunches  of  needle-shaped  crystals  of 
fatty  acids. 

False  or  pseudo-casts  are  not  infrequently  met  with  in 
urine  and  must  be  distinguished  from  the  preceding-  since 
they  have  an  entirely  different  origin.  Such  casts  may 
consist  of  mucous  threads,  or  of  mucous  threads  in  which 
urates  have  become  imbedded;  or  of  masses  of  bacteria 
(zo5g\lcea  threads).  Cholesterin  and  uric  acid  may  also 
occur  in  cast-like  form.  The  mucous  filaments  are  rather 
wide  and  often  branch,  and  are  insoluble  in  acetic  acid; 
hence  readily  distinguishable  from  hyaline  casts. 

Cylindroids  such  as  are  found  in  cholera,  scarlet  and 
recurrent  fever,  etc.,  resemble  somewhat  hyaline  casts. 
They  are  very  long-,  flat  bands  with  frayed  ends. 


Vegetable  Organisms. — Bacteria  are  not  present  in  nor- 
mal urine.  When  such  urine,  however,  is  collected  in 
ordinary  vessels  and,  moreover,  is  exposed  to  the  air, 
bacteria  are  soon  introduced  and  by  fcheir  activity  induce 
decomposition.  The  previously  clear  urine  becomes  cloudy 
due  to  the  suspended  bacteria  and  possibly  to  a  change  in 
the  reaction.  The  most  common  chang-e  thus  induced  is  the 
ammoniacal  fermentation  whereby  urea  undergoes  hydra- 
tion and  yields  ammonia  and  carbonic  acid.  The  power  of 
inducing  fermentation  of  this  type  belongs  not  to  one  species 


URINE.  207 

but  to  a  large  number  of  pathogenic  and  non-pathogenic 
bacteria.  Some  of  these  are  micrococci,  others  are  bacilli; 
sarcine  forms  may  be  present.  The  term  micrococcus  ureas, 
of  Pasteur,  does  not  denote  one  species  but  is  rather  to  be 
regarded  as  a  group  name  for  these  organisms.  No  clinical 
importance  is  attached  to  such  decompositions  when  they 
take  place  after  the  urine  has  been  passed.  It  not  infre- 
quently happens,  however,  that  bacteria  of  this  kind  have 
entered,  or  have  been  introduced  into  the  bladder  where 
they  grow  and  induce  exactly  the  same  change  as  that 
which  occurs  in  normal  urine  on  standing.  Such  urine,  con- 
taminated within  the  body,  will  be  cloudy  at  the  time  of 
passage  and  is  neutral  or  ammoniacal  in  reaction. 

Other  accidental  bacteria  introduced  in  a  similar  way 
may  induce  hydrogen  sulphide  fermentation  (hydrothio- 
nuria,  p.  196).  Such  urine  is  cloudy,  acid  in  reaction,  and 
has  the  odor  of  rotten  eggs. 

Certain  pathogenic  bacteria  may  be  present  in  urine 
and  their  recognition  is  consequently  a  matter  of  consider- 
able importance.  The  urine  is  centrifugated  and  the 
deposit  is  examined  according  to  the  methods  described  in 
text-books  on  bacteriology. 

Tubercle  bacilli  may  be  present  in  the  urine  in  tuber- 
cular affections  of  the  kidney,  bladder,  prostate,  etc. 
When  urine  persistently  contains  small  quantities  of  pus  or 
blood  it  should  be  repeatedly  examined  for  these  organisms. 
Care  should  be  taken  not  to  confound  the  smegma  bacillus 
with  the  tubercle  bacillus. 

The  gonococcus  ordinarily  will  be  found  in  the  first 
portion  of  urine  that  is  passed  since  this  disease  is  located 
in  the  urethra.  The  disease  may  extend  inward  so  that  the 
last  as  well  as  the  first  portion  of  the  urine  will  be  rich  in 
pus  corpuscles  and  in  gonococci. 

In  abscesses  of  the  kidney,  the  urine  may  contain 
staphylococci  and  streptococci.     In  severe  infections,  as  in 


208  PHYSIOLOGICAL  CHEMISTRY. 

typhoid  fever,  anthrax,  etc.,  the   specific  organisms  may 
appear  in  the  urine. 

The  necessity  of  using-  sterile  catheters  to  prevent 
infection  should  be  clearly  understood. 

Yeast  cells  are  common  in  diabetic  urines,  especially 
when  these  have  stood  for  some  time.  The  recognition  of 
yeast  cells  offers  no  difficulties. 

Moulds  may  develop  on  the  surface  of  urine  on  stand- 
ing. The  greenish  growth  of  penicillium  glaucum  is  most 
common.  The  fungus  of  lumpy-jaw,  actinomyces,  although 
not  strictly  belonging  under  this  head  may  be  mentioned 
as  having  been  found  in  urine. 

Animal  Organisms. — Parasitic  organisms  of  this  class 
are  not  infrequently  found  in  the  urine  in  tropical  countries. 
They  are  of  much  less  common  occurrence  in  the  higher 
latitudes,  and  even  in  such  cases  a  previous  residence  in 
a  warm  climate  may  often  be  established.  Infection  with 
these  animal  parasites  may  take  place  either  from  the 
blood,  through  the  kidneys,  or  from  the  exterior.  The  con- 
stant presence  of  a  little  blood  (tropical  hcematuria),  and  of 
small  aggregations  of  epithelial  cells  point  to  the  possible 
presence  of  such  organisms.  Some  of  the  so-called  worms 
found  in  urine  may  be  merely  altered  blood  clots. 

The  blood  may  bring  to  the  kidneys  echinococci,  the 
larval  form  of  the  tape- worm;  eggs  of  Distomum  haema- 
tobium; or  embryos  of  the  Filaria  sanguinis. 

Organisms  that  are  found  at  times  in  the  intestines  or 
in  the  vagina  may  also  be  met  with  in  urine.  Such  are  the 
Oxyuris  vermicularis,  Cercomonas  and  Trichomonas  vagin- 
alis. The  latter  has  been  found  but  a  few  times  in  urine, 
once  by  Dock. 


URINE.  209 

Unorganized  Sediment. 

Under  this  head  are  described  definite  chemical  com- 
pounds, which  may  either  be  amorphous  or  may  form  per- 
fect crystals.  They  are  unorganized  since  they  do  not 
possess  cell  structure.  These  chemical  substances  are 
usually  difficultly  soluble  acids,  or  difficultly  soluble  salts. 

Uric  acid  exists  in  the  urine  in  solution,  as  a  urate,  at  the 
time  of  passage.  On  cooling-,  as  a  result  of  mass  action,  the 
acid  phosphates  appropriate  the  base,  sodium,  and  hence 
free  uric  acid  crystallizes  out.  On  heating  the  urine  the 
precipitate  of  uric  acid  will  redissolve.  The  crystals  are 
invariably  reddish-yellow  or  brown  in  color  and  may-  var}' 
greatly  in  form.  They  may  be  identified  by  the  form  and 
color;  solubility  in  alkalis,  piperazin,  or  lysidin;  and  bj^ 
the  murexid  reaction. 

Urates  may  deposit  in  febrile,  or  in  concentrated  urines. 
They  are  usually  colored  pink  or  red  and  hence  the  deposit 
might  be  mistaken  for  blood.  On  heating  the  urine  the 
urates  promptly  dissolve.  On  the  addition  of  an  acid  to  the 
amorphous  urate  deposit  crystalline  uric  acid  forms.  The 
potassium  and  sodium  urates,  as  well  as  uric  acid,  are  met 
with  in  acid  urines.  In  neutral  or  ammoniacal  urine  the 
urate  of  ammonium  may  deposit  and  can  be  readily  recog- 
nized by  the  burr-like  form. 

In  order  to  apply  the  murexid  test  the  sediment  should 
be  removed  by  filtration,  washed  with  water,  then  with 
alcohol,  and  finally  transferred  to  a  dish  and  the  test 
applied  (Exp.  6,  p.  145). 

Hippuric  acid  is  very  rare  in  the  deposit  in  the  urine  of 
man.  It  deposits  under  similar  conditions  as  uric  acid.  The 
prismatic  crystals  likewise  color  a  yellowish-red  and  may 
be  mistaken  for  the  rarer  forms  of  uric  acid.     The  solubility 


210  PHYSIOLOGICAL  CHEMISTRY. 

in  alcohol,  etc.,  and  the  absence  of  the  murexid  reaction 
will  serve  to  distinguish  it  from  uric  acid. 

Cystin  forms  thin,  colorless  six-sided  plates.  It  is  a 
very  rare  constituent  and  may  give  rise  to  stones. 

Tyrosin  may  form  bundles  of  slender  needles.  Leucin 
is  more  likely  to  be  in  solution  but  on  concentration  it 
forms  characteristic  spherical  masses. 

Calcium  oxalate  may  appear  in  alkaline  as  well  as  acid 
urine.  It  usually  forms  characteristic  octahedra,  but  may 
exist  as  dumb-bells,  discs  or  may  even  be  amorphous.  The 
latter  can  be  distinguished  from  phosphates  and  urates  by 
its  behavior  to  acetic  acid  (insoluble) ;  it  is  soluble  in  hydro- 
chloric acid.  Oxalates,  in  small  numbers,  are  present  in 
normal  urine. 

Calcium  carbonate  is  especially  met  with  in  the  alkaline, 
cloudy  urine  of  herbivorous  animals.  It  occurs  amorphous, 
or  as  granules,  or  dumb-bells.  It  is  rare  in  the  urine  of 
man. 

Calcium  sulphate  is  likewise  a  rare  constituent  of  sedi- 
ments. It  forms  long,  colorless  prisms,  or  bunches  of 
needles  not  unlike  those  of  tyrosin.  It  is  insoluble  in 
acetic  acid  and  in  ammonia,  and  can  thus  be  distinguished 
from  tyrosin. 

Phosphates  are  deposited  usually  in  alkaline  urine.  If 
the  alkalinity  is  due  to  potassium  or  sodium  carbonate  the 
phosphates  of  calcium  and  of  magnesium  are  precipitated  in 
an  amorphous  form.  In  this  condition  they  are  readily 
washed  out  of  the  bladder.  If  the  urine  is  ammoniacal  the 
crystalline  ammonium  magnesium  phosphate,  commonly 
known  as  triple  phosphate,  will  deposit.  The  urine  may  be 
neutral  or  even  slightly  acid  and  yet  triple  phosphates  may 
form,  provided  some  ammonia  is  present.     The  crystalline 


URINE.  211 

form,  the  reaction  of  the  urine,  the  presence  of  ammon- 
ium urate  and  the  behavior  with  acetic  acid  will  enable 
identification. 

The  normal  magnesium  phosphate  may  occur  in  rhom- 
bic plates,  but  this  form  is  very  rare;  usually  it  is  amor- 
phous. The  acid  phosphate  of  calcium  may  likewise  form 
crystals  in  faintly  acid  urine. 

Cholesterin  crystals  may  float  on  the  surface  of  the  urine 
or  may  be  present  in  the  sediment.  The  same  is  true  of  fats 
and  fatty  crystals. 

Albumoses. — Certain  albumose  bodies  may  be  present  in 
the  sediment  in  amorphous,  or  in  crystalline  form.  Crys- 
talline proteins  are  very  rare  and  in  only  one  case  have 
they  been  observed  in  urine  (p.  186). 

Xanthin  crystals  are  likewise  exceedingly  rare.  They 
may  form  xanthin  stones. 

Indigo  may  deposit  as  dark  blue  stellate  needles  in 
alkaline  urine  which  is  rich  in  indox}d.  These  crystals 
may  float  on  the  surface  of  the  urine  or  may  be  found  in  the 
sediment. 

Bilirubin  has  been  met  with  in  acid  urine  as  amorphous 
yellow  granules  or  as  plates  imbedded  in  mucus.  It  is  a 
very  rare  constituent.  For  recognition  the  Gmelin  test  can 
be  applied  under  the  microscope. 

Urinary  Calculi. 

Calculi,  or  stones,  may  be  formed  in  the  kidneys  or 
in  the  bladder.  Any  one  of  the  numerous  substances 
which  may  be  present  in  the  sediment  may  enter  into  the 
composition  of  a  urinary  calculus.  If  the  urine  is  acid 
in  reaction  the  calculus  will  consist  of  substances  that 
dex>osit  in  acid  urine.     On  the  other  hand,  if  the  urine  is 


212  PHYSIOLOGICAL  CHEMISTRY. 

alkaline,  fixed  or  ammoniacal,  corresponding  compounds, 
such  as  neutral  phosphates,  or  triple  phosphate  will  be 
present  in  the  stone.  When  the  stone  is  formed  in  unde- 
composed  urine,  having-  an  acid,  or  fixed  alkaline  reaction, 
it  is  said  to  be  of  primary  formation.  But  when  organisms 
invade  the  bladder  and  set  up  ammoniacal  fermentation 
the  characteristic  deposit  of  ammonium  urate,  triple  phos- 
phate, etc.,  ensues.  The  stones  that  are  formed  in  this 
case  are  spoken  of  as  secondary. 

In  order  that  a  calculus  may  form,  whether  it  be  in  the 
kidney,  bladder,  liver  or  elsewhere,  it  is  necessary  that 
some  insoluble  substance  be  present  to  serve  as  a  nucleus, 
around  which  layer  after  layer,  or  crystal  on  crystal,  can 
be  deposited.  Just  as  a  crystal  of  sugar  or  piece  of  thread 
placed  in  a  thick  syrup  serves  to  start  the  crystallization 
or  deposition  of  the  sugar,  so  a  crystal  of  uric  acid,  or  of 
triple  phosphate,  or  a  piece  of  fat  may  serve  as  the  start- 
ing point  in  the  formation  of  a  urinary  calculus.  Uric  acid 
and  urates  are  by  far  the  most  common  substances  which 
form  the  nuclei  of  stones — about  81  per  cent.  Less  than  9 
per  cent,  of  the  calculi  begin  with  a  nucleus  of  earthy 
phosphates,  and  less  than  6  per  cent,  begin  with  one  of 
calcium  oxalate.  Cases  are  known  where  tallow  or  paraffin 
introduced  by  catheters,  or  by  injection,  constituted  the 
nucleus.  In  other  instances,  blood  clots,  or  mucous  threads 
served  the  same  purpose.  Usually  there  is  but  one  nucleus 
in  a  stone,  although  two  or  more  may  be  present. 

Urinary  calculi  may  be  divided  into  simple  and  com- 
posite. The  simple  stones  are  rare  and  consist  of  but  one 
chemical  substance.  Thus,  we  may  have  calcium  oxalate, 
cystin,  xanthin,  fat,  or  cholesterin  stones. 

The  composite  stones  are  much  more  common.  In 
these  there  are  usually  several  substances  arranged  in 
layers.  Thus,  a  layer  of  uric  acid  may  be  followed  by  one 
of  urates,  this  by  phosphates,  oxalates,  and  the  like.    Varia- 


URINE.  213 

tion  in  the  reaction  of  the  urine  will  bring  about  different 
deposits,  and  may  even  result  in  the  removal  of  substances 
already  deposited.  Thus,  a  uric  acid  layer  may  be  followed 
by  earthy  phosphates,  or  may  even  be  replaced  by  these,  if 
the  urine  possesses  fixed  alkaline  reaction.  If  subsequently 
ammoniacal  decomposition  sets  in  triple  phosphate,  am- 
monium urate,  etc.,  may  be  thrown  down. 

Stones  may  vary  in  size  from  that  of  a  pea  to  that  of 
an  egg.  When  the  accretions  are  small  and  numerous  they 
are  passed  with  the  urine  and  are  spoken  of  as  gravel. 

Uric  acid  calculi  are  by  far  the  most  common  and  con- 
sist either  of  the  free  acid  or  of  urates.  They  are  invaria- 
bly colored  yellowish  to  dark  red.  The  surface  may  be 
smooth  or  rough,  and  when  broken  a  concentric  arrange- 
ment may  usually  be  seen.  The  uric  acid  may  alternate 
with  oxalates. 

Ammonium  urate  stones  are  smaller,  softer  and  more 
crumbling  than  the  preceding.  They  are  rare  as  primary 
stones  (in  children);  common  as  secondary  stones. 

Calcium  oxalate  yields  perhaps  the  hardest  stones. 
They  are  distinctly  crystalline  and  not  infrequently  the 
octahedral  crystals  can  be  made  out  by  the  eye.  The 
sharp  pointed  ends  of  these  crystals  impart  a  very  rough 
surface  to  the  almost  colorless  stone  and  may  cause  haem- 
orrhages and  severe  pain  if  passed  by  the  urethra. 

Phosphatic  calculi  are  commonly  secondary  and  are  mix- 
tures of  phosphates  (normal  and  triple),  carbonates, 
urates  and  oxalates.  They  may  attain  considerable  size. 
In  color  they  are  usually  white,  gray  or  yellow  and  they 
have  a  more  or  less  chalky  character.  Simple  stones  of 
triple  phosphate,  or  of  acid  phosphate  of  calcium,  are  rare. 

Calcium  carbonate  stones  are  rare  in  man  but  common  in 
herbivorous  animals. 


214  PHYSIOLOGICAL    CHEMISTRY. 

Gystin  calculi  are  likewise  very  rare,  occurring-  only  in 
cystinuria.  They  have  a  pale  yellow  color  and  a  smooth, 
soft  surface.  They  are  primary  in  origin  and  may  attain 
the  size  of  an  egg. 

Xanthin  stones  are  exceedingly  rare  and  vary  greatly 
in  size.  They  are  usually  brownish  in  color  and  on  rubbing 
they  take  on  a  waxy  appearance. 

Urostealiths. — This  term  is  applied  to  very  rare  calculi 
which  consist  almost  entirely  of  fat  or  rather  of  fatty 
acids,  either  free,  or  combined  with  calcium  and  magne- 
sium.    They  are  light,  soft  and  have  a  brownish  color. 

Calculi  of  fibrin  or  of  coagulated  blood,  and  of  mucus 
have  also  been  met  with. 

Cholesterin  stones. — Although  these  are  common  in  bile, 
they  are  extremely  rare  in  urine.  Indeed,  only  one  instance 
is  known.     The  same  is  true  of  Indigo  calculi. 


Examination  of  Urinary  Calculi. 

The  following  method  of  analysis  taken  from  Ham- 
marsten  will  serve  as  a  guide  in  such  examinations.  If 
the  stone  shows  distinct  layers,  each  one  of  these  should 
be  tested  by  itself: 

Heat  a  portion  of  the  powdered  stone  on  a  platinum  foil. 

A.     It  does  not  burn. 

The  original  powder  treated  with  HC1. 

(a).     Effervesces. — Calcium  carbonate. 

(b).     Does  not  effervesce.      A   portion  of  the   powder  is  gently- 
ignited  and  then  is  treated  with  HC1. 

(a).    Effervesces. — Calcium  oxalate. 


URINE.  215 

(6).     Does  not  effervesce.     The  original  powder  is  warmed 
with  potassium  hydrate  solution. 

1. — Ammonia  is  freely  given  off.  The  powder  dissolves  in 
acetic  or  hydrochloric  acid,  and  this  solution  with  am- 
monia gives  a  crystalline  precipitate. — Triple  phos- 
phate. 
2. — Ammonia  is  not  given  off,  or  but  in  traces.  The  pow- 
der dissolves  as  above  and  the  solution  with  ammonia 
yields  an  amorphous  precipitate. — Earthy  phosphates. 
B.     It  does  burn. 

(a).     With  a  flame. 

1.— The  flame  is  yellow,  lasting;  odor  of  burnt  feathers;  insol- 
uble in  ether  and  alcohol. — Fibrin. 
2. — The  flame  is  yellow,   lasting;   odor  of  burning  resin  or 

shellac;  soluble  in  ether  and  alcohol. — Urostealith. 
3. — The  flame  is  bluish;  does  not  last;    peculiar  sharp  odor; 
soluble  in  ammonia  from  which  solution  on  spontaneous 
evaporation  six-sided  plates  separate. — Cystin. 
(6).     Without  a  flame. 

1. — Does  not  give  the  murexid  test;  dissolves  in  HN03  without 
effervescence,  and  this  solution  on  evaporation  leaves  a 
yellow  residue  which  with  alkalis  turns  orange,  on  heating 
becomes  red. — Xanthin. 
2.— Does  give    the    murexid    test.      The  original  powder  is 
treated  with  a  little  cold  KOH  solution. 
(a).     Ammonia  is  freely  given  off. — Ammonium  urate, 
(b).    Ammonia  is  not  given  off,  or  but  in  traces. — Uric  acidt 


216 


PHYSIOLOGICAL  CHEMISTRY. 


Table  of  Atomic  Weights. 


(ACCORDING  TO  F.  W.  CLARKE.) 


Name. 

Symbol. 

Atomic 
weight. 

Name. 

Symbol. 

Atomic 
weight. 

Antimony 

Arsenic 

Barium 

Al 

Sb 

As 

Ba 

Bi 

B 

Br 

Cd 

Cs 

Ca 

C 

Ce 

CI 

Cr 

Co 

Cb 

Cu 

Di 

Er 

F 

Ga 

Ge 

Gl 

Au 

H 

In 

I 

Ir 

Fe 

La 

Pb 

Li 

Mg 

Mn 

Hg 

27 
120 

75 
137 
208.9 

11 

79.95 
112 
132.9 

40 

12 
140.2 

35.45 

52.1 

59 

94 

63.6 
142.3 
166.3 

19 

69 

72.3 

9 

197.3 

1.007 
113.7 
126.85 
193.1 

56 

138.2 
206.95 
7.02 

24.3 

55 
200 

Molybdenum  . . . 
Nickel 

Mo 

Ni 

N 

Os 

O 

Pd 

P 

Pt 

K 

Rh 

Rb 

Ru 

Sm 

Sc 

Se 

Si 

Ag 

Na 

Sr 

S 

Ta 

Te 

Tb 

Tl 

Th 

Sn 

Ti 

W 

U 

V 

Yb 

Yt 

Zn 

Zr 

96 

58.7 

Osmium 

Oxygen 

14.03 

190.8 

Bismuth 

16 

Boron 

Palladium 

Phosphorus 

Platinum 

Potassium 

Rhodium 

Rubidium 

Ruthenium 

Samarium 

Scandium 

Selenium 

Silicon 

106.6 

Bromin 

31 

Cesium 

195 
39.11 

Carbon 

103 

85.5 

Cerium 

101.6 

Chlorin 

150 

Cobalt 

44 

79 

Columbium 

28.4 

Copper 

Silver 

107.92 

Erbium 

Sodium 

23.05 

Sulphur 

Tantalum 

Tellurium 

Terbium 

Thallium 

Thorium 

Tin 

87.6 

Fluorin 

32.6 

Gallium 

Germanium  .... 

Gold 

182.6 
125 
159.5 
204.18 

Hydrogen 

Indium 

232.6 
119 

Iodin 

Titanium 

Vanadium 

Ytterbium 

Zinc 

48 

Iridium 

184 

Iron 

239.6 

Lanthanum  .... 
Lead 

51.4    - 
173 

89.1 
65.3 

Manganese 

Mercury 

Zirconium. ...... 

90.6 

CHAPTER     XL 
QUANTITATIVE  ANALYSIS. 

Quantitative  analysis  can  be  carried  out  in  two  ways: 
gravimetrically  or  by  weight,  and  volumetrically  or  by  meas- 
ure. 

In  gravimetric  determinations  the  substance  to  be  esti- 
mated is  converted  into  an  insoluble  form  which  is  then . 
removed  by  filtration,  washed  free  of  all  impurities,  dried 
and  weighed.  In  some  instances  the  precipitate  is  not 
weighed  directly  but  is  first  ignited  and  thus  converted 
into  a  more  stable  form.  As  an  illustration  we  may  men- 
tion the  estimation  of  albumin  in  urine.  This  is  converted 
into  an  insoluble  form  by  the  action  of  heat;  the  coagulated 
albumin  is  collected  on  a  previously  weighed  filter,  washed, 
dried  and  weighed.  After  deducting  the  weight  of  the 
dried  filter,  the  difference  represents  the  weight  of  albumin 
present  in  the  quantity  of  urine  taken  for  the  examination. 
Usually  substances  cannot  be  weighed  directly,  as  such,  but 
must  be  combined  with  other  metals  or  acids  to  form  insol- 
uble compounds.  Thus,  if  it  is  desired  to  estimate  the 
chlorine  present  in  urine  the  most  convenient  procedure  is 
to  precipitate  the  chlorides  present  with  silver  nitrate. 
The  silver  chloride  is  then  collected,  washed,  dried,  ignited 
and  weighed.  From  the  amount  of  silver  chloride  found  it 
is  easy  to  calculate  the  corresponding  amount  of  CI,  or  of 
NaCl.  Similarly,  when  sulphuric  acid  is  estimated,  advan- 
tage is  taken  of  the  fact  that  it  forms  an  insoluble  com- 
pound with  barium  chloride.  The  barium  sulphate  is  col- 
lected on  a  filter,  washed,  dried,  ignited  and  weighed,  and 
from  the  weight  obtained  the  amount  of  S03  present  can  be 
calculated. 


218  PHYSIOLOGICAL  CHEMISTRY. 

The  ignition  serves  to  destroy  the  filter  and  in  some 
cases  also  alters  the  composition  of  the  precipitate.  Thus, 
calcium  oxalate  on  ignition  is  converted  into  calcium  oxide; 
magnesium  ammonium  phosphate  is  changed  to  the  pyro- 
phosphate. The  filter  on  incineration  leaves  a  certain 
amount  of  ash,  the  weight  of  which  should  be  known,  as 
well  as  that  of  the  crucible  in  which  the  ignition  takes 
place;  these  weights  must  therefore  be  deducted  from  the 
final  weight  in  order  to  obtain  the  weight  of  the  pre- 
cipitate. 

When  a  precipitate  is  filtered  off,  the  filtrate  should 
come  through  perfectly  clear.  If  jt  does  not  do  so  it  should 
be  returned  to  the  filter  until  it  does  come  through  clear, 
otherwise  a  loss  of  the  precipitate  would  result.  Further- 
more, great  care  must  be  taken  to  wash  the  precipitate  on  the 
filter  till  all  the  substances  that  may  be  present  in  solution 
are  washed  out  of  the  filter  and  precipitate.  If  for  instance 
chlorides  are  being  estimated,  the  silver  chloride  is  washed 
on  the  filter  till  a  portion  of  the  wash-water  ceases  to  give 
the  slightest  cloud  on  addition  of  HC1.  This  indicates  that 
all  the  silver  nitrate  has  been  removed  by  the  washing. 

In  volumetric  analysis  the  amount  of  the  substance 
present  is  ascertained  by  means  of  solutions  of  known 
strength.  These  are  spoken  of  as  standard  and  are  of  two 
kinds:  empirical  and  normal. 

An  empirical  solution  is  of  such  strength  that  one  c.c. 
will  react  with  exactly  so  many  mg.  of  the  substance  to 
be  analyzed  as  may  be  desired.  In  other  words  one  c.c.  of 
an  empirical  solution  indicates  1,  5,  10,  or  any  number  of 
mg.  of  that  substance  according  to  its  strength.  It  may  be 
made  to  represent  any  number  of  milligrams  of  such  sub- 
stance as  may  be  convenient  for  purpose  of  calculation. 
Thus,  we  may  make  several  empirical  solutions  of  silver 


QUANTITATIVE  ANALYSIS.  219 

nitrate:  one  c.c.  of  one  solution  may  represent  1  mg.  of  CI; 
that  of  another  10  mg.  of  01;  that  of  a  third  10  mg-.  of  NaCl, 
etc. 

To  prepare  an  empirical  solution  write  out  first  of  all 
the  equation  representing  the  reaction  between  the  reagent 
and  the  substance  to  be  analyzed.  Thus,  we  will  prepare 
an  empirical  solution  of  AgN03  such  that  one  c.c.  will  ex- 
actly precipitate  all  the  chlorine  contained  in  10  mg.  of 
NaCl.  In  other  words  one  c.c.  is  to  represent  10  mg.  of 
NaCl. 

AgNOs  +  NaCl  =  AgCl  +  NaNO,. 
170  58.5 

It  is  evident  that  one  molecule  of  AgN03  will  react 
with  one  of  NaCl,  and  since  their  molecular  weights  are  170 
and  58.5  respectively  it  follows  that  170  g.  of  silver  nitrate 
will  react  with  58.5  g.  of  sodium  chloride.  The  question 
then  is,  how  much  AgN03  will  react  with  .010  g.  of  NaCl? 
This  is  easily  found  by  the  following  proportion: 


Mol.  weight  of 

Mol.  weight  of 

AgNOs 

NaCl 

170 

:         58.5    :  :    x  :  0.010 

x  =  0.02906  g.  AgNOs. 

That  amount  of  AgN03  must  be  present  in  one  c.c.  in 
order  that  this  shall  react  with  10  mg.  of  NaCl.  Therefore, 
29.060  g.  of  AgN03  dissolved  in  water  and  diluted  to  one 
liter  will  give  an  empirical  solution  such  that  one  c.c. 
represents  10  mg.  of  NaCl. 

Calculate  the  amount  of  reagent  necessary  to  make  a 
liter  of  empirical  solution  of  each  of  the  following: 

AgN03,  such  that  one  c.c.  will  represent  10  mg-.  of  CI; 

"  "  "  "  2mg.  ofKCl. 

BaCl2  "  "  "  "  10  mg.  of  SOs; 

"  "  "  "  "  10  mg.  of  Na2SQ4. 


220  PHYSIOLOGICAL  CHEMISTRY. 

How  much  Hg,  HgO,  Hg(N03)2  must  be  dissolved  and 
diluted  to  one  liter  in  order  to  make  an  empirical  solution 
such  that  one  c.c.  will  represent  10  mg.  of  urea? 

A  normal  solution  is  one  that  contains  in  one  liter  one 
gram  of  basic  hydrogen  or  its  equivalent.  Thus,  in  HC1 
the  hydrogen  is  basic;  moreover  one  part  of  hydrogen 
unites  with  35.5  parts  of  chlorine  to  make  36.5  parts  of  HC1. 
In  other  words  1  g.  of  basic  hydrogen  is  contained  in  36. 5  g. 
of  HC1.  This  amount  of  HC1,  dissolved  and  diluted  to  one 
liter,  will  give  therefore  a  normal  solution  of  HC1  (N). 

Again,  H2S04  =  2  +  32  +  64  =  98.  That  is,  in  98  g.  of 
H2S04  there  are  2  g.  of  basic  hydrogen.  The  definition 
above  calls  for  one  g.  of  basic  hydrogen,  hence  49  g.  of 
H2S04  dissolved  and  diluted  to  one  liter  would  give  a 
normal  H2S04  solution. 

Calculate  the  amount  necessary  to  make  one  liter  of 
normal  solution  of  each  of  the  following:  HN03,  H3P04, 
anhydrous  oxalic  acid  C2H204,  crystallized  oxalic  acid 
C2H204  +  2  H20,  acetic  acid  C2H402.  In  calculating  these 
bear  in  mind  that  a  normal  solution  does  not  call  for  1  g.  of 
hydrogen  but  for  1  g.  of  basic  hydrogen. 

The  calculation  for  a  normal  alkali  solution,  such  as 
sodium  hydrate,  is  as  follows: 

NaOH  +  HC1  =  NaCl  +  H20. 
40         36.5 

40  g.  of  sodium  hydrate,  then,  combine  with  36.5  g.  of 
hydrochloric  acid  containing  one  gram  of  basic  hydrogen. 
Therefore  40  g.  NaOH  contain  the  equivalent  of  one  gram 
of  basic  hydrogen,  and  when  dissolved  and  diluted  to  one 
liter  make  a  normal  solution. 

Calculate  how  much  sodium  carbonate  (Na2C03)  is  con- 
tained in  a  normal  solution.     Also  calculate  the  amounts 


QUANTITATIVE   ANALYSIS.  221 

of   KOH,  Ba(OH)2,  Ag20  respectively  that  must  be  taken 
and  dissolved  to  make  a  liter  of  normal  solution. 

The  normal  solutions  are  too  strong  for  most  purposes, 
hence  solutions  of  fractional  strength  are  employed.  Thus, 
a  semi-normal  solution  of  hydrochloric  acid,  1  HC1,  is  one 
which  contains  18.25  g.  HC1  per  liter.  A  deci-  or  tenth- 
normal solution  (ftr)  contains  one-tenth  and  a  centi-normal 
solution  (ttsu)  contains  one-hundredth  the  amount  that  a  nor- 
mal one  does. 

As  a  rule  deci-normal  solutions  are  most  convenient 
and  are  therefore  employed  most  often. 

One  c.c.  of  any  normal  acid  solution  will  exactly  neu- 
tralize 1  c.c.  of  any  normal  alkali  solution.  This,  follows 
from  the  definition  of  such  solution.  Thus,  36.5  g.  of  HC1 
neutralize  40  g.  of  NaOH.  The  one  contains  one  gram  of 
basic  hydrogen,  the  other  its  equivalent.  Since  these 
amounts  are  each  contained  in  one  liter  of  corresponding 
normal  solutions,  it  follows  that  a  liter  of  one  will  neutral- 
ize a  liter  of  the  other;  or  one  c.c.  of  one  will  neutralize  one 
c.c.  of  the  other. 

Furthermore,  1  c.c.  of  a  normal  solution  corresponds  to 
10  c.c.  of  a  deci-normal  solution;  1  c.c.  of  a  deci-normal 
solution  corresponds  to  1  c.c.  of  any  other  deci-normal 
solution.  The  same  is  true  of  other  fractional  normal 
solutions. 

The  word  factor  means  the  amount  of  the  reagent 
contained  in  1  c.c.  of  the  solution.  Thus,  the  N  factor  of 
NaOH  is  0.040;  the  ft  factor  of  hydrochloric  acid  is  0.00365. 
When  speaking  of  empirical  solutions  the  word  factor 
means  the  amount  of  the  substance  to  be  analyzed  repre- 
sented by  one  c.c.  of  that  solution. 

The  term  titration  is  employed  to  denote  the  method  of 
estimating  a  given  substance  in  solution  by  means  of  a 
standard  solution.     Thus,  we  can  estimate  the  amount  of 


222  PHYSIOLOGICAL   CHEMISTRY. 

chlorides  in  urine  by  titration  with  a  standard  solution  of 
silver  nitrate. 

Indicators. — For  the  purpose  of  determining  when  a 
reaction  is  completed  an  indicator  is,  as  a  rule,  necessary. 
An  indicator  usually  shows  by  a  change  in  the  color  of  the 
solution  when  enough  of  the  standard  solution  has  been 
added.  Thus,  in  titrating  an  alkaline  solution,  with  litmus 
as  an  indicator,  the  moment  a  red  color  appears  we  know 
that  the  reaction  has  changed  from  alkaline  to  acid,  and 
that  enough  of  the  standard  acid  solution  has  been  added. 

In  preparing  standard  solutions  of  such  reagents  as  do 
not  change  when  [exposed  to  air,  the  required  amount  can 
be  weighed  directly,  then  dissolved  and  diluted  to  one  liter. 
This  is  true  of  such  substances  as  silver  nitrate,  copper 
sulphate,  oxalic  acid,  etc., — substances  which  are  crystal- 
line and  neither  absorb  moisture  nor  give  off  water  of  crys- 
tallization when  exposed.  But  the  case  is  different  with 
such  substances  as  NaOH  which  rapidly  absorb  moisture 
and  carbon  dioxide,  or  with  the  common  acids  which  have 
a  variable  strength.  In  such  cases,  it  is  customary  to 
dissolve  a  little  more  than  the  required  amount  of  the 
reagent,  then  determine  the  strength  of  this  solution  gravi- 
metrically,  or  volumetrically.  The  preparation  of  xrr  NaOH 
as  given  on  p.  239  will  illustrate  this  point. 

When  a  substance  contains  water  of  crystallization  this 
must  be  taken  into  account  in  the  calculation  since  it  is  for 
the  time  being  a  part  of  the  molecule,  and  although  the 
water  plays  no  part  in  the  reaction  it  nevertheless  weighs 
something. 

A  good  balance  is  necessary  for  quantitative  analysis. 
Certain  rules  regarding  the  use  of  the  balance  should  be 
closely  adhered  to.  The  first  thing  to  do  is  to  see  that  the 
scales  balance,  that  is,  that  the  pointer  swings  the  same 
number  of  divisions  on  each  side  of  the  zero  point.     The 


QUANTITATIVE  ANALYSIS.  223 

first,  or  starting-  swing  is  disregarded,  the  second  and  third 
are,  however,  noted.  If  the  scales  do  not  balance  they 
should  be  made  to  do  so,  either  by  cleaning  the  watch- 
glasses  and  pans,  or  by  turning  the  adjustment. 

The  substance  is  weighed  either  on  the  watch-glass  or 
in  a  weighing-bottle,  never  on  the  pan  direct.  The  sub- 
stance should  always  be  placed  on  the  left  pan;  the  weights 
on  the  right  pan.  Whenever  a  portion  of  the  substance,  or 
a  weight  is  to  be  placed  on,  or  taken  off  the  balance,  the 
latter  should  first  be  brought  to  a  perfect  rest. 

The  weights  should  never  be  handled  with  the  fingers, 
but  should  always  be  picked  up  with  a  pair  of  clean  forceps. 
When  weighing  the  weights  should  not  be  placed  on  the  pan 
at  random,  guessing  that  this  or  that  much  will  be  enough, 
but  they  should  be  placed  on  in  regular  order.  Thus,  for 
example,  in  weighing-  a  crucible  it  is  found  that  10  g\  is  too 
much.  This  is  taken  off  and  5  g.  placed  on;  as  this  is  too 
light  the  2  g.  piece  is  added;  this  also  is  insufficient  and  so  a 
1  g.  piece  is  added.  This  is  still  not  enough  so  another  1  g. 
weight  is  placed  on  the  pan.  The  total  weight  of  9  g.  is 
still  less  than  that  of  the  crucible,  which,  however,  does 
not  weigh  10  g.  The  500  mg.  piece  is  then  placed  on  the 
pan,  but  the  total  weight  is  now  too  heavy;  the  crucible 
weighs  less  than  9.5  g.  The  200  mg.  piece  is  substituted 
but  this  is  likewise  too  heavy;  then  a  100  mg.  piece  is 
tried.  This  is  too  light;  the  weight  of  the  crucible  lies 
between  9.1  and  9.2.  Now  the  50  mg.  piece  is  added;  this 
is  not  heavy  enough,  and  so  the  20  mg.  piece  is  placed  on; 
this  is  still  too  light  and  a  10  mg.  piece  is  added ;  as  this  is 
not  enough  the  second  10  mg.  piece  is  added;  but  even  now 
the  weight  is  low.  It  lies,  therefore,  between  9.190  and  9.2. 
The  rider  is  now  placed  at  5,  or  in  its  absence  the  5  mg. 
piece  is  added.  If  the  5  mg.  piece  is  too  heavy  proceed 
exactly  as  above  with  the  500  mg.  piece;  if  it  is  not  heavy 
enough  proceed  as  with  the  50  mg.  piece.  When  weighing 
with  less  than  the  10  mg.  piece,  or  when  using  the  rider, 


224  PHYSIOLOGICAL  CHEMISTRY. 

the  door  of  the  balance  should  be  closed.  The  balance 
should  be  sensitive  to  one  milligram  and  a  good  one  should 
read  to  iV  mg. 

To  measure  out  liquids  the  graduated  cylinder,  pipette, 
or  burette  is  made  use  of.  The  cylinder  is  not  very  accur- 
ate and  should  not  be  used  except  to  measure  out  roughly 
10,  25,  50  c.c.,  etc.  Each  student  is  provided  with  a  pipette 
and  a  burette,  holding  10  and  50  c.c.  respectively,  and  gra- 
duated in  tV  c.c.  These  are  to  be  used  for  measuring  out 
solutions  for  exact  work.  The  surface  of  the  liquid  in  the 
pipette  or  burette  is  concave,  forming  the  so-called  menis- 
cus. When  taking  a  reading  the  eye  should  be  placed  on  a 
level  with  the  lower  border  of  the  meniscus  since  this  is 
more  distinct  than  the  upper. 

If  the  measuring  instrument  is  not  perfectly  dry  it 
should  be  washed  with  distilled  water,  then  rinsed  two  or 
three  times  with  small  portions  of  the  liquid  that  is  to  be 
measured.  The  outside  of  the  pipette  or  burette  should  be 
wiped  dry.  When  using  the  burette  care  must  be  taken  to 
expel  any  air  that  may  remain  in  the  tip.  If  the  pipette  or 
burette  is  dirty,  that  is,  if  water  does  not  flow  readity  and 
smoothly  but  adheres  in  drops,  here  and  there,  it  can  be 
cleaned  by  filling  with  the  chromic  acid  cleaning  mixture 
and  allowing  it  to  stand  thus  over  night. 

In  gravimetric  work  the  amount  of  ash  given  by  the 
filter  paper  on  ignition  should  be  known.  It  is  best  to  use 
filters  washed  in  HC1  and  HF.  The  ash  from  these  is  so 
small  that  it  may  be  disregarded.  The  paper  should  be 
folded  carefully,  and  unless  a  dry  filter  is  called  for,  it 
should  be  moistened.  The  tip  of  the  funnel  should  not  be  so 
far  above  the  surface  of  the  filtrate  in  the  beaker  as  to 
cause  spattering.  The  filter  should  never  be  filled  too  full, 
since  many  precipitates  tend  to  creep  up  and  a  portion  may 
thus  be  lost.     A  glass  rod  covered  with  a  short  piece  of 


QUANTITATIVE    ANALYSIS.  225 

rubber  tubing-,  y2  inch  long,  is  of  great  use  in  transferring 
precipitates  from  the  beaker  or  flask  to  the  filter. 

The  filtrate  should  always  be  received  in  a  clean  beaker 
or  flask,  and  the  first  portion  should  be  tested  to  ascertain 
if  the  precipitation  is  complete  by  adding  some  of  the 
reagent  used.  In  washing,  small  amounts  of  wash-water 
should  be  used,  and  each  portion  should  be  allowed  to  run 
through  before  another  is  added.  If  the  liquid  does  not 
filter  clear,  the  filtrate  should  be  returned  to  the  filter  a 
second,  or  even  third  time  if  necessary. 

The  funnel  with  the  filter  containing  the  precipitate  is 
placed  in  an  air-bath  at  100°  to  dry.  It  should  be  covered 
with  paper. 

Everything  that  is  set  aside  should  be  properly  labelled, 
and  covered  to  protect  it  from  dust.  The  weighings  and 
other  results  should  be  recorded  at  once  in  a  note-book,  and 
not  kept  on  scraps  of  paper,  or  trusted  to  memory. 

Quantity  of  Urine. 

The  quantity  of  urine  passed  during  a  period  of 
twenty-four  hours  is  of  great  importance  both  as  an  indi- 
cation of  the  presence  or  absence  of  certain  diseases,  as 
well  as  the  basis  of  computations  in  the  quantitative  analy- 
sis of  this  secretion.  During  a  period  of  twenty-four  hours 
there  are  hourly  variations  in  the  volume  of  urine  and  in 
the  amount  of  solids  present,  and  consequently  analytical 
results  can  have  but  little  value  unless  obtained  from  a  fair 
sample  of  the  entire  day's  urine. 

In  collecting  the  urine  care  should  be  taken  to  empty 
the  bladder  before  entering  upon  and  again  at  the  close  of 
the  24  hour  period.  Furthermore,  loss  of  urine  at  stool 
should  be  avoided.  Urine  should  always  be  received  in 
perfectly  clean  vessels,  care  being  taken  to  avoid  the  acci- 
dental introduction  of   fats,  oils,  starches,  fibers  and  the 


226  PHYSIOLOGICAL  CHEMISTRY. 

like.  The  urine  may  be  measured  in  ounces,  pints  or  quarts, 
but  it  is  much  better  to  procure  a  metric  graduate  express- 
ing- the  volume  in  so  many  cubic  centimeters.  It  may  be 
well  to  bear  in  mind  that  a  quart  is  a  little  less  than  a  liter. 

An  adult  man  excretes  on  an  average  about  1500  c.c.  of 
urine  per  day;  women  excrete  less,  about  1200  c.c.  This 
difference  in  volume  corresponds  to  a  like  difference  in  the 
amounts  of  urea,  uric  acid,  etc.,  excreted,  and  is  due  to  the 
different  conditions  under  which  the  two  sexes  live  rather 
than  to  any  difference  of  sex  proper.  The  average  volume 
of  the  urine  of  333  male  students  at  Ann  Arbor  was  1120  c.c. , 
and  the  average  specific  gravity  for  the  same  number  was 
1.022.  The  average  volume  of  urine  of  56  female  students 
was  1000  c.c,  and  the  average  specific  gravity  was  1.020. 
Infants  excrete,  relatively,  3 — 4  times  as  much  urine  as 
adults.  Children,  likewise,  excrete  relatively  more,  where- 
as in  old  age  the  excretion  is  less. 

Great  variations  in  the  quantity  of  urine  may  be  ex- 
pected in  different  individuals  and  even  in  the  same  indi- 
vidual, depending  on  certain  conditions  mentioned  below. 
Regardless  of  the  variation  in  the  volume  of  the  urine  of  a 
healthy  person  it  can  be  said  that  the  solids  in  solution 
remain  constant,  whether  the  volume  is  great  or  small.  In 
diseased  conditions,  on  the  other  hand,  variations  in  volume 
and  in  solids  will  be  met  with.  The  least  secretion  of  urine 
takes  place  during  the  night,  toward  morning;  whereas 
greatest  secretion  is  observed  after  rising  and  especially 
an  hour  or  two  after  meals. 

When  the  volume  of  urine  is  materially  increased  this 
condition  is  designated  as  polyuria.  A  diminished  secretion 
of  urine  is  known  as  oliguria,  and  suppression  of  urine  secre- 
tion is  expressed  by  the  term  anuria. 

There  are  certain  factors  which  markedly  influence  the 
volume  of  urine  in  health  and  in  disease.  These  may  be 
briefly  touched  upon. 


QUANTITATIVE   ANALYSIS.  227 

1. — The  amount  of  water  ingested;  whether  taken  as 
such,  or  as  milk,  coffee,  tea,  beer  and  the  like,  it  promptly 
increases  the  volume  of  urine.  It  should,  moreover,  be 
remembered  that  animal  and  vegetable  food  contains 
nearly  three-quarters  of  its  weight  of  water.  This  diuretic 
action  of  water  can  be  readily  utilized  whenever  it  is  nec- 
essary or  desirable  to  flush  out  the  kidneys. 

2. — The  kind  and  amount  of  food  is  to  be  considered. 
As  just  indicated  a  large  percentage  of  ordinary  food  is 
water.  The  percentage  is  still  greater  (87  per  cent)  in  the 
case  of  milk.  Moreover,  the  hydrogen  in  the  starch,  fat,  or 
proteid  molecule  is  largely  oxidized  in  the  body  to  water, 
and  hence  it  is  that  more  urine  may  be  excreted  than  the 
amount  of  water  actually  consumed.  Furthermore,  in  star- 
vation, urine  continues  to  be  excreted  even  when  no  water 
is  taken.  This  is  due  to  the  setting  free  of  water  held  by 
the  tissues  when  they  are  broken  down,  and  to  the  forma- 
tion of  water  by  the  oxidation  of  the  hydrogen  of  such 
disintegrated  tissue.  In  general,  the  tissue  thus  broken 
down  will  yield  about  three-fourths  of  its  weight  of  water. 
Certain  constituents  of  food  by  their  diuretic  action  increase 
the  volume  of  urine.  This  is  true  of  salt,  onions,  coffee, 
tea,  etc. 

3. — The  amount  of  water  that  leaves  the  body  other- 
wise than  by  the  kidneys.  These  other  channels  of  elimin- 
ation are  the  skin,  lungs,  stomach  and  intestines.  In 
addition  to  these  water  may  be  diverted  from  the  kidneys  to 
form  exudates  or  transsudates,  as  in  oedema  and  in  ascites. 
A  warm  temperature  and  much  exercise  favor  the  loss  of 
water  by  perspiration  and  by  exhalation.  Thus,  after  much 
walking  or  wheeling  in  summer  time  the  amount  of  urine  is 
small  (several  hundred  c.c.)  although  an  enormous  amount 
of  water  may  be  drunk.  Such  urine  has  a  high  specific 
gravity  and  is  highly  colored.  Prolonged  vomiting  may 
serve  to  remove  water  and  with  this,  injurious  waste  pro- 
ducts.    In  intestinal  diseases,  especially  where  a  diarrhoeic 


228  PHYSIOLOGICAL  CHEMISTRY. 

condition  exists  as  in  cholera,  enormous  loss  of  water  may- 
occur  through  the  discharges  and  as  a  result  the  quantity 
of  urine  will  be  diminished  and  even  complete  anuria  may- 
result. 

4. — Nervous  influences  may  affect  the  volume  of  urine, 
as  in  hysteria;  possibly  also  in  diabetes  insipidus. 

5. — Diseases  of  the  kidney,  heart,  lungs,  etc. 

An  increased  excretion  of  urine  is  met  with  in  diabetes 
insipidus,  in  which  condition  the  volume  of  urine  may  be 
several  times  that  of  the  normal,  and  inasmuch  as  there  is 
no  increase  in  solids  the  specific  gravity  may  be  very  low 
and  the  color  very  light. 

In  diabetes  mellitus,  contrary  to  other  diseases  or  con- 
ditions, the  specific  gravity  of  the  urine  (1.030 — 1.040  or 
more)  increases  with  the  increase  in  the  volume  of  the 
urine.  A  diabetic  person  on  an  average  eliminates  5  to  6 
liters  of  urine  per  day,  but  it  is  not  unusual  to  find  double 
this  amount.  As  a  rule  the  more  water  that  is  excreted, 
the  more  sugar  there  is  present  in  the  urine  and  hence  the 
increase  in  specific  gravity.  This  great  increase  of  urine 
in  a  diabetic  person  may  be  counteracted  by  a  febrile  dis- 
ease, in  which  case  the  volume  may  be  diminished,  though 
not  always,  to  less  than  the  normal  volume.  The  urine 
is  increased  in  icterus  after  the  obstruction  in  the  bile  duct 
has  been  removed. 

A  decrease  in  the  volume  of  urine  is  of  considerable 
importance  and  is  met  with  in  various  diseases.  In  dys- 
pnceic  conditions,  due  to  disturbances  of  the  circulatory  or 
respiratory  apparatus,  the  urine  is  diminished  in  volume; 
the  specific  gravity  is  increased  and  the  color  is  deepened; 
it  is  strongly  acid  in  reaction,  and  urate  sediments  are  com- 
mon. This  is  due  to  loss  of  water  by  the  lungs,  skin,  and 
by  transsudates. 

In  febrile  diseases,  until  crisis  occurs,  the  volume  of  the 


QUANTITATIVE  ANALYSIS.  229 

urine  is  small,  the  specific  gravity  is  high  and  the  urine 
possesses  the  characteristics  just  mentioned.  In  a  person 
having  contracted  kidneys,  or  affected  with  diabetes,  the 
volume  of  the  urine  is  not  diminished,  as  a  rule,  below  that 
of  normal  urine.  When  crisis  occurs,  as  in  pneumonia, 
scarlet  fever,  etc.,  there  is  at  once  a  marked  increase  in 
volume,  the  specific  gravity  drops  and  the  color  becomes 
lighter.  In  recovery  from  protracted  fever,  as  typhoid,  the 
urine  gradually  returns  to  normal  volume,  specific  gravity 
and  color. 

In  rheumatism  the  urine  is  small  in  amount,  with 
high  specific  gravity  and  a  sediment  as  in  fevers.  Toward 
the  end  of  the  attack  the  urine  is  increased  in  amount  and 
the  specific  gravity  decreases.  In  starvation,  with  or 
without  water  being  taken,  there  is  a  decrease  in  the  vol- 
ume of  urine.  A  fasting  person  does  not  drink  much  water, 
and  moreover  the  loss  of  water  by  the  lungs  and  skin 
continues. 

In  diseases  of  the  liver  the  amount  of  urine  varies  con- 
siderably, depending  on  the  amount  of  water  ingested  and 
whether  or  not  ascites  exists.  In  stomach  diseases  the 
volume  may  be  greatly  diminished,  depending  on  the 
amount  of  food  taken,  and  the  extent  of  absorption.  The 
latter  may  be  diminished  by  vomiting  or  otherwise,  and 
hence  the  urine  will  be  small  in  volume.  In  kidney  dis- 
eases the  volume  of  the  urine  may  vary  from  complete 
anuria  to  marked  polyuria. 


I. — Determination  of  the  Quantity  of  Urine  per  24  Hours. 


(a).     By  volume. — Cylinders  graduated  in  cubic  centimeters  should 
be  employed. 

(6).     By  weight. — This  is  more  accurate  but  is  less  commonly  used.. 


230  PHYSIOLOGICAL  CHEMISTRY. 


Specific  Gravity  of  Urine. 

The  specific  gravity  of  urine  depends  upon  the  volume 
and  on  the  amount  of  solids  held  in  solution.  The  density 
of  normal  urine  consequently  varies  considerably,  but  as 
a  rule  it  lies  between  1.017  and  1.020.  As  a  result  of 
drinking-  much  water  it  may  fall  to  1.010  or  1.005,  whereas 
when  very  little  water  is  taken  it  may  rise  to  1.030.  As 
stated  above,  in  a  healthy  person  the  total  amount  of  solids 
excreted  is  quite  constant,  from  day  to  day,  regardless  of 
the  quantity  of  the  urine.  This  great  variation  in  the 
specific  gravity  of  normal  urine  depends  almost  wholly  on 
the  amount  of  water  taken  into  the  body  and  excreted  by 
the  kidneys.  If  considerable  water  is  lost  by  perspiration  or 
by  exhalation,  as  after  much  exercise  in  warm  weather,  the 
volume  of  the  urine,  as  already  pointed  out,  will  be  greatly 
decreased  and,  as  there  is  no  difference  in  the  excretion  of 
solids,  it  follows  that  the  specific  gravity  will  be  increased. 
The  solids  which  especially  affect  the  specific  gravity, 
since  they  make  up  two-thirds  of  the  total  amount  present, 
are  urea  and  sodium  chloride. 

The  determination  of  the  specific  gravity  of  urine  is 
one  of  the  most  important  steps  taken  in  the  analysis  of 
urine.  It  'gives  valuable  information  as  to  the  extent  of 
tissue  metabolism  and  the  efficiency  of  the  kidneys.  It  is 
necessary,  however,  to  always  bear  in  mind  not  only  the 
amount  of  water  ingested  but  also  the  amount  of  water 
that  leaves  the  body  by  other  channels  than  the  kidneys, 
such  as  by  perspiration,  exhalation,  exudation,  vomiting 
and  diarrhoea.  Likewise  the  kind  and  amount  of  food 
taken  must  be  considered. 

A  high  specific  gravity  and  a  small  amount  of  urine  are 
met  with  especially  in  acute  febrile  diseases.  Although 
such  persons  take  little  or  no  food,  the  amount  of  urea,  as 


QUANTITATIVE    ANALYSIS.  231 

has  been  pointed  out  (p.  129),  is  considerably  increased. 
Furthermore,  the  amount  of  water  taken  in  is  small  and 
a  considerable  amount  is  lost  through  other  channels  than 
the  kidneys.  A  high  specific  gravity,  therefore,  indicates 
increased  tissue  destruction.  In  convalescence  there  is 
less  tissue  destruction,  hence  less  urea  is  formed,  and  this 
with  the  increased  quantity  of  urine  gives  a  low  specific 
gravity. 

A  high  specific  gravity  and  a  small  amount  of  urine  are 
met  with  in  many  mental  disorders,  such  as  delirium,  and  in 
all  diseases  which  lead  to  venous  stasis.  This  is  due  to  the 
decrease  in  the  elimination  of  water.  Furthermore,  the 
very  highest  specific  gravity  of  urine  (1.040 — 1.050)  is  met 
with  frequently  in  diseases  of  the  kidney,  especially  in  the 
later  stages.  This  is  due  to  the  presence  of  albumin  and 
to  the  retention  of  water.  In  the  early  stages  of  kidney 
diseases,  however,  the  urine  is  considerably  increased  in 
quantity  and  although  albumin  is  present,  the  specific 
gravity  is  low. 

A  low  specific  gravity  is  met  with  in  all  conditions 
which  increase  the  quantity  of  the  urine,  except  in  dia- 
betes mellitus.  Such  conditions  are  convalescence,  early 
stages  of  kidney  diseases,  and  diabetes  insipidus  (1.010  or 
less). 

A  low  specific  gravity  and  a  small  amount  of  urine  are 
observed  in  many  chronic  diseases,  and  just  before  death 
from  acute  diseases.  It  indicates  slow  metabolism,  or 
retention  and  points  to  danger  from  uraemia. 

A  high  specific  gravity  and  a  large  volume  of  urine  are 
met  with  in  only  one  disease,  diabetes  mellitus.  The  in- 
creased density,  in  spite  of  the  enormous  amount  of  water 
passed,  is  due  to  the  large  amounts  of  sugar,  urea,  etc., 
which  are  excreted.  This  condition  of  the  urine  is  therefore 
a  strong  indication  of   the  presence  in  the  urine  of   sugar 


232  PHYSIOLOGICAL    CHEMISTRY. 

and  the  latter  should  at  once  be  tested  for.  On  the  other 
hand,  a  low  specific  gravity  and  a  large  amount  of  urine 
should  lead  to  the  suspicion  of  albuminuria,  and  tests  for 
albumin  should  be  made. 


II.     Determination  of  the  Specific  Gravity. 

(a).  With  the  urinometer. — The  spindle  of  this  instrument  is  grad- 
uated from  1.000  to  1.040  at  a  temperature  of  15°  C.  Hence,  if  the 
temperature  is  higher,  as  is  usually  the  case,  the  observed  reading 
should  be  reduced  to  the  normal  temperature  of  15°. 

Pour  the  urine  to  be  tested  into  a  cylinder  which  should  be  wide 
enough  to  allow  the  urinometer  free  motion.  Avoid  the  formation  of 
foam  and  if  any  results  remove  it  by  means  of  a  piece  of  filter  paper. 
Immerse  the  clean  dry  urinometer  and  after  it  comes  to  rest  read  off 
the  point  where  the  lower  border  of  the  meniscus  cuts  the  scale.  To 
make  sure  that  the  instrument  floats  freely  gently  touch  the  stem 
of  the  urinometer  and  after  it  comes  to  rest  take  a  second  reading. 
The  normal  specific  gravity  varies  from  1.017 — 1.020.     (See  p.  230). 

To  reduce  the  observed  reading  to  the  normal  temperature,  take 
the  temperature  of  the  urine  and  for  every  3°  above  15°  add  one 
division,  and  for  every  3°  below  subtract  one  division  from  the  ob- 
served reading.     Thus: 

The  specific  gravity  of  a  urine  is  1.017  and  the  temperature  is 
24°.     24  —  15  =  9.     Therefore,  1.017  +  3  =  1.020,  the  correct  reading. 

If  a  sediment  of  urates  is  present  in  the  urine  this 
should  be  gently  warmed  till  they  are  dissolved.  Other- 
wise it  is  desirable,  though  not  necessary,  to  filter  the 
urine.  In  order  to  draw  accurate  conclusions,  the  specific 
gravity  should  not  be  taken  of  a  portion  of  the  urine 
passed  at  one  time  but  of  the  mixed  24  hours'  urine. 

The  urinometer  offers  the  quickest  means  for  taking 
the  density  of  a  urine.  The  results  obtained  are  very 
satisfactory,  especially  if  the  correction  is  made  for  tem- 
perature as  indicated  above,  and  if  a  set  of  two  or  of  four 
urinometers  is  used.  The  ordinary  urinometer  is  gradu- 
ated from  1.000  to  1.040  and  consequently  each  degree  is 


QUANTITATIVE    ANALYSIS.  233 

so  small  that  an  error  in  reading-  may  easily  occur.  This 
error  can  be  obviated  by  using  a  set  of  two  urinometers, 
one  of  which  is  graduated  from  1.000  to  1.020;  the  other 
from  1.020  to  1.040.  Better  still  is  a  set  of  four  urinometers 
graduated  as  follows:  from  1.000  to  1.010;  1.010  to  1.020; 
1.020  to  1.030;  and  1.030  to  1.040.  With  the  latter  set  it  is 
possible  to  read  to  the  fourth  decimal  place.  Whereas,  on 
the  ordinary  urinometer  the  40  divisions  are  crowded  into 
about  3  cm.,  in  the  set  mentioned  above  they  extend  over 
about  20  cm.,  hence  the  greater  accuracy  in  reading. 

Reduction  of  the  observed  specific  gravity  to  that  corresponding 
to  the  normal  volume  of  1500  c.c:  multiply  the  quantity  of  urine 
per  24  hours  by  the  decimal  portion  of  the  specific  gravity,  divide  by 
1500  and  add  the  result  to  1.  Thus,  the  volume  of  a  urine  is  1820 
c.c,  and  the  specific  gravity  is  1.014. 

1820  X  .014 

1.  H =  1.017. 

1500 

Again,  the  volume  is  4500  c.c.  and  the  specific  gravity  is  1.030. 
Therefore: 

4500  X  .030 

1.  -\ =  1.090. 

1500. 

(b).  With  the  picnometer,  or  specific  gravity  bottle.  This  is  the 
most  accurate  means  for  ascertaining  the  specific  gravity  but  it  is 
employed  only  for  very  exact  work. 

III.     Determination  of  Total  Solids. 


a).  Neubav&r's  approximate  method.— Multiply  the  last  two  figures 
of  the  specific  gravity  by  2.33;  this  gives  the  weight  of  solids  in  1000 
c.c.  To  obtain  the  solids  in  the  24  hours'  urine  multiply  this  result 
by  the  number  of  liters  of  urine. 

Examples.— The  volume  of  the  urine  is  1820  c.c  and  the  specific 
gravity  is  1.014.  14  X  2.33  X  1.820  =  59.37  g.  of  solids  in  the  24  hours' 
urine. 


234  PHYSIOLOGICAL    CHEMISTRY. 

Again,  the  volume  is  4500  c.c.  and  the  specific  gravity  is  1.030. 
30  X  2.33  X  4.500  =  314.55  g.  of  solids  per  24  hours'  urine. 

(&).  Direct  determination. — This  is  rarely  resorted  to 
except  for  scientific  purposes.  Inasmuch  as  a  part  of  the 
urea  is  decomposed,  during-  the  process  of  evaporation,  into 
ammonia  and  carbonic  acid,  the  amount  of  ammonia  given 
off  must  be  determined,  and  the  result  converted  into 
urea.  The  amount  of  urea  thus  ascertained  to  be  broken 
up  by  the  evaporation  of  a  known  volume  of  the  urine 
on  the  water-bath  is  added  to  the  weight  of  the  residue 
obtained. 

The  amount  of  total  solids  present  in  normal  urine  is 
quite  constant,  regardless  of  variation  in  the  volume  of  the 
urine.  The  average  amount  of  solids  is  about  60  g.  The 
organic  compounds  make  up  about  35  g.  and  the  inorganic 
compounds  about  25  g.  Urea  is  the  chief  organic  compound 
and  amounts  to  about  30  g.  Common  salt  is  the  chief  inor- 
ganic constituent  and  makes  up  about  15  g.  For  the  quanti- 
ties of  other  constituents  present  see  the  table  at  the  end 
of  this  chapter. 

The  urinometer  gives  the  specific  gravity  of  the  urine, 
and  from  this  the  amount  of  total  solids  can  be  estimated. 
In  a  somewhat  similar  manner  the  amount  of  urea,  of  sugar 
and  of  albumin  may  be  estimated  from  the  readings  of  the 
urinometer. 

Reaction  of  Urine. 

The  reaction  of  normal  urine  is  usually  acid,  due  to  the 
presence  of  the  acid  phosphate  of  potassium  or  sodium, 
NaH2P04.  The  presence  of  Na2HPO+  decreases  the  acidity 
and  may  even  render  the  urine  neutral  or  amphoteric  in 
reaction.  It  has  always  been  an  interesting  question  as  to 
how  an  acid  urine  is  secreted  from  an  alkaline  blood.  The 
reaction  of  blood,  as    seen  from  previous  experiments,  is 


QUANTITATIVE    ANALYSIS.  235 

strongly  alkaline,  due  to  alkaline  carbonates  and  phos- 
phates. It  has  been  supposed  that  the  absorption  of  the  free 
hydrochloric  acid  from  the  stomach  and  the  formation  of 
sulphuric  and  phosphoric  acids  by  the  oxidation  in  the  body 
of  sulphur  and  phosphorus  containing-  substances,  led  to 
the  formation  of  acid  phosphates  in  the  blood.  These  acid 
salts,  as  is  well  known,  can  dialyze  more  readily  than 
alkaline  phosphates  through  an  animal  membrane,  and 
hence  would  appear  in  the  urine  and  by  their  preponderance 
impart  the  characteristic  reaction  to  the  secretion  (Maly). 
According  to  Liebermann  the  formation  of  acid  phosphates 
takes  place  in  the  renal  epithelial  cells.  These  were  found 
to  contain  a  compound  of  lecithin  and  albumin,  lecith- 
albumin,  which  possessed  marked  acid  properties.  The 
alkaline  urates  and  phosphates,  consequently,  come  in 
contact  with  this  lecith-albumin  which  takes  up  an  atom  of 
sodium  from  each  and  leaves  acid  urates  and  acid  phos- 
phates which  then  pass  out  into  the  urine. 

A  similar  instance  of  mass  action  is  seen  in  the  change 
in  reaction  that  frequently  takes  place  in  urine  on  standing. 
At  the  time  of  passage  it  may  be  intensely  acid,  but  on 
cooling  and  standing  it  becomes  less  acid,  and  even  neutral, 
and  free  uric  acid  appears  in  the  sediment.  By  virtue  of 
mass  action,  assisted  by  the  low  temperature,  the  acid 
phosphates  remove  the  alkali  from  the  uric  acid  and  thus 
set  this  compound  free.  At  the  same  time  they  form  neutral 
phosphates  and  hence  the  change  in  the  reaction  of  the 
urine.  When  the  temperature  is  raised  the  uric  acid  can 
regain  the  lost  atom  of  sodium  and  hence  passes  into  solu- 
tion as  an  urate  and  at  the  same  time  the  original  acid 
reaction  returns. 

An  acid  fermentation  of  urine  may  take  place  and  thus 
the  acidity  may  be  considerably  increased.  Thus,  in  hydro- 
gen sulphide  fermentation  the  reaction  will  be  intensely 
acid  (p.  196).     Again,  if  sugar  is  present  this  may  undergo 


236  PHYSIOLOGICAL  CHEMISTRY. 

lactic  or  butyric  acid  fermentation.  The  cause  of  such 
acid  fermentations  is  invariably  one  or  more  species  of 
bacteria. 

The  urine  may  be  alkaline  due  either  to  sodium  or  to 
potassium  carbonate  {fixed  alkali),  or  to  ammonia  or  am- 
monium carbonate  {volatile  alkali)  (p.  136).  When  due  to 
fixed  alkali  the  cause  is  invariably  to  be  found  in  the 
food.  As  pointed  out  under  urea  (p.  126)  the  salts  of  most 
organic  acids  are  oxidized  to  the  corresponding'  carbonates. 
Thus,  potassium  acetate,  citrate,  malate,  etc.,  are  each 
converted  into  potassium  carbonate  and  this  is  then 
excreted  by  the  urine.  It  is  possible,  therefore,  by  direct 
administration  of  such  salts  to  convert  a  highly  acid  urine 
into  one  having"  less  acidity,  or  even  an  alkaline  reaction. 
On  the  other  hand  many  articles  of  food,  as  fruits  and  pota- 
toes, contain  such  organic  salts  and  hence  impart  an  alka- 
line reaction  to  the  urine. 

An  alkaline  reaction  due  to  volatile  alkali  is  practically 
met  with  only  in  ammoniacal  fermentation.  All  urine, 
sooner  or  later,  undergoes  ammoniacal  fermentation,  the 
urea  becoming  hydrated  to  ammonia  and  carbonic  acid. 
However,  it  is  only  when  this  change  occurs  in  the  bladder, 
when  the  urine  at  the  time  of  passage  has  an  ammoniacal 
reaction,  that  it  possesses  a  pathological  significance.  The 
cause  of  ammoniacal  fermentation  is  always  a  micro-organ- 
ism introduced  in  some  way  from  without  the  body  (see 
p.  137). 

While  the  reaction  of  the  mixed,  twenty-four  hours'  nor- 
mal urine  is  usually  acid,  this  is  not  the  case  with  the 
several  portions  passed  during  that  period.  There  may  be 
hourly  variations  in  the  reaction  as  well  as  in  the  quantity 
and  composition  of  the  urine  secreted.  Thus,  the  urine 
secreted  during  digestion  (3-6  hours  after  meals)  may  be 
neutral,  or  even  temporarily  alkaline,  due  to  the  secretion 
of  a  large  amount  of  hydrochloric  acid  into  the  stomach. 


QUANTITATIVE    ANALYSIS.  237 

The  more  hydrochloric  acid  secreted  into  the  gastric  juice 
the  more  marked  will  be  the  decrease  in  the  acidity  of  the 
urine.  In  diseases  of  the  stomach,  such  as  cancer,  where 
little  or  no  acid  is  secreted  it  follows  that  there  will  be  no 
effect  observed  on  the  acidity  of  the  urine.  The  morning- 
urine  is  often  neutral  or  alkaline  in  reaction,  whereas  that 
secreted  in  the  afternoon  or  early  evening-  is  the  most  acid. 

From  what  has  been  said  it  is  evident  that  the  food  is 
of  first  importance  as  affecting  the  reaction  of  the  urine. 
This  is  seen  in  the  reaction  of  the  urine  of  carnivorous  as 
compared  with  that  of  herbivorous  animals.  The  former 
is  strongly  acid,  the  latter  neutral  or  alkaline.  In  the 
former  case  the  protein  matter  of  the  food,  rich  in  sulphur 
and  phosphorus,  is  oxidized  in  the  body  and  yields  sulphuric 
and  phosphoric  acids.  The  food  of  herbivorous  animals 
on  the  other  hand  is  rich  in  organic  salts,  which  are  oxidized 
in  the  body  to  carbonates.  It  does  not  follow  that  all 
vegetable  food  will  impart  an  alkaline  reaction.  If  such 
food  is  rich  in  proteins,  as  in  the  case  of  cereals  and  legu- 
mens,  it  will  yield  almost  as  acid  a  urine  as  a  meat  food. 
Potatoes,  fruit  and  the  like  are  poor  in  proteins  and  rela- 
tively rich  in  organic  salts,  hence  they  impart  an  alkaline 
reaction.  In  starvation  the  urine  is  acid,  owing  to  the 
fac^  that  the  animal  is  living  on  the  proteins  of  its  own 
tissue^. 

^The  influence  of  digestion  on  the  reaction  of  urine  has 
been  indicated  above.  Muscular  exercise  is  said  to  increase, 
and  profuse  perspiration  is  said  to  decrease  the  acidity. 
The  absorption  of  alkaline  transudates  may  render  the 
urine  alkaline. 

While  it  is  true  that  an  exclusively  meat  diet  produces 
a  strongly  acid  urine  due  to  the  sulphuric  and  phosphoric 
acids  formed  on  oxidation,  it  does  not  follow  that  the  ad- 
ministration of  these  acids  will  enable  one  to  increase  the 
acidity  of  the  urine  without  limit.  When  the  alkalis  of 
the  blood  cannot  be  spared  or  are  not  sufficient  in  amount  to 


238  PHYSIOLOGICAL  CHEMISTRY. 

combine  with  the  acid  administered  the  latter  unites  with 
the  ammonia  which  normally  would  go  to  make  urea,  and 
appears  in  the  urine  as  an  ammonium  salt.  This  actually 
takes  place  in  the  urine  of  man  and  of  carnivorous  animals. 
In  herbivorous  animals,  however,  it  would  seem  as  if  the 
ammonia  formed  in  protein  disintegration  was  not  at  the 
ready  disposal  of  the  administered  mineral  acids  and  hence 
cannot  neutralize  and  render  harmless  such  acids.  The 
continued  administration  of  mineral  acids  in  small  doses  to 
herbivorous  animals  results  in  the  withdrawal  or  in  the 
decrease  of  the  alkaline  carbonates  of  the  blood  to  such 
an  extent  as  to  cause  death. 

The  reaction  of  the  urine,  and  hence  diet,  has  special 
significance  in  connection  with  the  formation  of  uric  acid 
sediments  and  calculi.  Food  rich  in  proteins  and  poor  in 
the  bases  necessary  to  combine  with  the  sulphuric  and 
phosphoric  acids  formed  on  oxidation  should  be  avoided. 
Cheese,  salted  meat  and  fish  are  of  this  class  and  when 
used  as  a  common  article  of  food  stones  are  frequent.  The 
deposition  of  urate  sediments  can  be  largely  controlled  by 
the  use  of  such  food  as  fruits,  potatoes,  etc.,  which  decrease 
the  acidity  of  urine  and  in  themselves  do  not  furnish  uric 
acid.  The  use  of  lithia  waters,  piperazin  or  lysidin  regard- 
less of  the  kind  of  food,  can  impart  but  little  benefit. 

Ammoniacal  urine,  because  of  the  crystalline  triple 
phosphate  that  deposits,  is  much  more  likely  to  cause  the 
formation  of  calculi  than  a  fixed  alkaline  urine.  The  fine, 
amorphous  phosphates  formed  in  the  latter  instance  are 
easily  washed  out.  Moreover,  this  reaction  is  usually  tem- 
porary and  is  due  to  the  food  and  can  therefore  be  readily 
corrected. 


QUANTITATIVE    ANALYSIS.  239 

IV.     Determination    of  the  Acidity  (or  Alkalinity)  of  the 

Urine. 

Reagents. —  ^  NaOH  solution.  This  contains  4  g.  of  NaOH  in  one 
liter:  therefore  each  c.c.  =  0.004  g.  of  NaOH.  Inasmuch  as  NaOH 
readily  absorbs  C02  and  water  from  the  air,  it  cannot  be  weighed  out 
accurately  enough  for  this  purpose.  Hence,  weigh  out  about  3  g.  and 
dissolve  in  about  500  c.c.  of  water.  This  solution  is  now  too  strong 
and  its  strength  must  therefore  be  ascertained;  the  solution  can  then 
be  diluted  to  the  proper  point. 

i1^  Oxalic  acicl  solution. — This  contains  6.3  g.  of  oxalic  acid  (H2C204 
+  2  H20)  in  one  liter;  hence  each  c.c.  =  0.0063  g.  of  oxalic  acid.  Oxalic 
acid  is  not  altered  on  exposure  to  the  air  and  can  therefore  be 
weighed  out  directly. 

Preliminary  exercise  in  titration.— Place  10  c.c.  of  the  oxalic  acid 
solution  in  a  small  beaker,  dilute  with  about  50  c.c.  of  water,  add  a  few 
drops  of  phenol-phthalein  solution  to  serve  as  indicator,  and  then  add 
from  a  burette,  drop  by  drop,  stirring  after  each  addition  with  a 
glass  rod,  the  sodium  hydrate  solution  until  a  faint  but  permanent 
pink  color  remains.  The  difference  in  the  reading  of  the  burette  be- 
fore and  at  the  close  of  the  titration  gives  the  amount  of  sodium 
hydrate  solution  employed  to  neutralize  10  c.c.   of  j\  oxalic  acid. 

Read  off  the  volume  of  NaOH  solution  remaining  in  the  cylinder 
and  calculate  the  amount  of  water  that  must  be  added  to  make  this 
strictly  deci-normal.  Thus,  suppose  that  the  10  c.c.  of  ^  oxalic  acid 
required  9.2  c.c.  of  the  NaOH  solution  for  neutralization,  and  that 
440  c.c.  of  NaOH  solution  remain  in  the  cylinder: 

9.2:  10::  440:  x.         x  =  478  c.c, 

the  volume  to  which  the  NaOH  should  be  diluted  in  order  that  it 
shall  be  deci-normal. 

Now  repeat  the  titration  with  the  properly  diluted  NaOH  solu- 
tion. If  correct,  10  c.c.  of  the  one  should  neutralize  10  c.c.  of  the 
other. 

Application  to  tht  wrine. — Owing  to  the  color  of  the  urine  and  the 
presence  of  certain  organic  substances  phenol-phthalein  cannot,  un- 
fortunately, be  used  and  in  its  stead  the  less  sensitive  litmus  papers 
are  employed.     Mix  well  the  24  hours'  urine  and   if    any  urates   are 


240  PHYSIOLOGICAL  CHEMISTRY. 

present  dissolve  them  by  the  aid  of  gentle  heat;  measure  out  100  c.c. 
into  a  beaker  and  from  a  burette  run  in  -fa  NaOH,  \%  c.c.  at  a  time, 
stirring"  after  each  addition.  After  each  addition  test  the  reaction 
with  litmus  papers  and  note  the  point  where  the  reaction  changes 
from  acid  to  neutral  or  slightly  alkaline.  The  latter  will  more  likely 
be  the  case.  Suppose  that  on  the  addition  of  3  c.c.  the  reaction 
was  still  acid  but  when  3.5  c.c.  were  added  it  became  slightly  alkaline. 
Therefore  the  neutral  point  lies  between  3  and  3.5  c.c. 

To  determine  exactly  the  point  of  neutralization,  measure  out 
a  fresh  portion  of  urine,  as  above,  and  add  3  ex.  of  fa  NaOH.  Then 
run  in  this  reagent,  drop  by  drop,  stiring  after  each  addition,  and 
also  testing  the  reaction  after  each  addition  with  litmus  papers  till 
the  neutral  point  is  reached.  When  this  is  reached  note  the  amount 
of  fa  NaOH  used.  Ascertain  by  calculation  the  amount  necessary  to 
neutralize  the  total  24  hours'  urine. 

Example. — Suppose  100  c.c.  of  the  urine  requires  3.4  c.c.  t^NaOH 
and  the  24  hours'  urine  amounts  to  1250  c.c. 

100  :  3.4  ::  1250  :  x.     x  =  42.5  c.c.  fa  NaOH. 

Results  can  be  expressed  in  this  way  but  it  is  better  to  convert 
the  number  of  c.c.  of  fa  NaOH  into  the  corresponding  amount  of 
oxalic  acid.  Each  c.c.  of  fa  NaOH  =  0.0063  g.  of  oxalic  acid.  There- 
fore, 42.5  X  0.0063  =  0.26775  g.  of  oxalic  acid.  That  is  to  say,  the 
acidity  of  the  24  hours'  urine  corresponds  to  that  much  oxalic  acid. 

If  the  urine  is  alkaline  the  degree  of  alkalinity  can  be  ascer- 
tained by  titrating  with  fa  oxalic  acid.  Or,  a  known  amount  of  fa^ 
oxalic  acid,  sufficient  to  produce  an  acid  reaction,  may  be  added  and 
the  liquid  then  titrated  back  to  the  neutral  point  with  fa  NaOH.  The 
difference  between  the  amount  of  oxalic  acid  added  and  that  left 
uncombined  corresponds  to  the  alkalinity  of  the  urine.  The  result 
is  converted  into  the  corresponding  amount  of  sodium  hydrate.  The 
results  obtained  by  this  method  are  not  very  exact,  owing  to  the 
presence  toward  the  end  of  the  reaction  of  alkaline  as  well  as  acid 
phosphates  and  hence  an  amphoteric  reaction  may  be  manifest. 


QUANTITATIVE    ANALYSIS.  241 


V.     Determination  of  Chlorides 


{a).    Volhard's  Method. 

Beaqents: — 

1. — Silver  nitrate  solution. — Dissolve  29.060  g.  of  fused  silver  nitrate 
in  one  liter  of  water.     Each  c.c.  =  10  mg.  of  sodium  chloride. 

2. — A  cold  saturated  solution  of  ferric  alum,  or  a  5  per  cent,  solution 
of  ferric  sulphate.  This  solution  must  be  free  from  chlorides.  It  is 
used  as  an  indicator. 

3. — Potassium  sulphocyanate  solution. — Dissolve  about  10  g.  of  the 
salt  in  a  ^  liter  of  water  and  standardize  the  solution  against  the 
silver  nitrate.  This  is  done  as  follows:  To  10  c.c.  of  the  silver  nitrate 
in  a  small  beaker,  diluted  to  about  50  c.c,  add  a  few  drops  of  the  ferric 
solution  and  then  nitric  acid,  drop  by  drop,  till  the  mixture  is  colorless. 
Now  run  in  from  a  burette  the  sulphocyanate  solution,  stirring-  well 
after  each  addition,  till  a  permanent  red  color  is  obtained.  Note  the 
amount  used.  10  c.c.  of  one  should  equal  10  c.c.  of  the  other.  If  the 
sulphocyanate  is  too  strong-  it  must  be  diluted  so  that  the  two  solu- 
tions have  the  same  strength. 

Example. — 450  c.c.  of  the  sulphocyanate  solution  is  left  in  the 
cylinder;  10  c.c.  of  the  silver  nitrate  solution  require  8.2  c.c.  of 
the  former.  10  —  8.2  =  1.8.  That  is,  to  every  8.2  c.c.  of  the  sulpho- 
cyanate 1.8  c.c.  water  must  be  added. 

Therefore,  8.2  :  10  ::  450  :  x  =  548.8. 

The  450  c.c.  in  the  cylinder  diluted  to  548.8  c.c.  give  a  solution 
of  the  proper  strength. 

Execution. — In  a  flask  provided  with  a  100  c.c.  mark  place  10  c.c. 
of  urine,  20—30  drops  of  dilute  nitric  acid  (1.185  sp.  gravity)  then 
several  drops  of  the  ferric  solution.  Now  run  in  from  a  burette  the 
silver  solution  till  the  precipitate  ceases  to  form.  Add  a  few  c.c. 
more  of  the  reagent.  Note  the  total  amount  used.  Fill  up  to  the 
100  c.c.  mark  with  water,  mix  and  filter  through  a  dry  filter.  To  50 
c.c.  of  the  filtrate  run  in  from  a  burette  the  sulphocyanate  solution, 
stirring  well,  till  a  faint  but  permanent  red  tinge  results.  Note  the 
amount  of  sulphocyanate  used,  multiply  it  by  two  and  deduct  this 
from  the  amount  of  silver  nitrate  used.     The  difference  is  the  amount 


242  PHYSIOLOGICAL  CHEMISTRY. 

of   silver  nitrate  solution  which  combined  with  the  chlorine.     Now 
calculate  the  amount  of  NaCl  in  the  24  hours'  urine. 

Example. — The  24  hours'  urine  amounts  to  1250  c.c,  and  to  10  c.c. 
of  it  17  c.c.  of  the  silver  solution  were  added.  50  c.c.  of  the  filtrate 
required  1.5  c.c.  of  the  sulphocyanate  solution;  hence  the  entire 
filtrate  would  require  2X1-5  =  3  c.c.  Therefore,  that  much  silver 
nitrate  solution  has  been  added  in  excess  above  that  necessary  to 
unite  with  the  NaCl  present  in  10  c.c.  of  the  urine. 

17  —  3  —  14  c.c,  the  amount  of  silver  nitrate  solution  actually 
used  up  by  the  NaCl  present.  Since  each  c.c.  of  silver  solution 
equals  10  mg.  of  NaCl,  14  c.c.  equals  14  X  10  =  140  mg.  =  0.140  g.  NaCL 

10  :  0.140  ::  1250  :  x.        x  =  17.50  g.  of  NaCl  in  the  24  hours'  urine. 
(6).    Mohr's  Method.  ' 

Reagents: — 

1. — Silver  nitrate  solution  of  the  same  strength  as  that  used  in 
Volhard's  method. 

2. — A  saturated  solution  of  yellow  potassium  chromate  (K2Cr04).  This 
must  be  free  from  chlorine  and  serves  as  an  indicator.  ^^ 

Execution. — Place  10  c.c.  of  the  urine  in  an  evaporating  dish,  add 
2  or  3  drops  of  nitric  acid,  about  100  c.c.  of  water,  and  2 — 3  drops 
of  the  potassium  chromate  solution.  Now  add  from  a  burette  the 
silver  nitrate  solution  until  a  faint  red  tinge  appears.  This  is  due  to 
the  formation  of  silver  chromate  and  shows  that  all  the  chlorine  has 
been  precipitated  and  an  excess  of  silver  added.  Note  the  amount 
of  silver  solution  used,  deduct  1  c.c.  and  multiply  the  difference  by 
10;  this  gives  the  number  of  mg.  of  sodium  chloride  in  10  c.c.  of  urine. 
Calculate  the  amount  present  in  the  24  hours'  urine. 

This  method  is  more  convenient  than  the  preceding  but  gives 
higher  results.  For  that  reason  0.5  —  1  c.c.  is  deducted  from  the 
observed  reading  in  case  the  urine  is  strongly  colored.  If  it  is  an 
aqueous  solution  that  is  titrated,  as  in  the  case  of  an  unknown,  the 
correction  should  be  disregarded.  Albumin  if  present  should  be  re- 
moved according  to  the  method  given  under  sugar. 

(c).  Gravimetric  Method. — Place  a  definite  amount  of  the 
solution  C10  c.c.  of  urine,  or  10  c.c.  of  the  unknown)  in  a  small  beaker, 
dilute  to  about  150  c.c.  and  acidify  with  2 — 3  c.c.  of  HN03.  Then  add 
silver  nitrate  solution  in  slight  excess  and  heat  to  boiling  for  a  few 
minutes.      Filter,    and    wash  the    precipitate   first  with  hot  water 


QUANTITATIVE    ANALYSIS.  243 

slightly  acidified  with  HN03,  then  with  cold  water  till  free  from 
silver  salts.  This  point  is  ascertained  by  collecting-  a  little  of  the 
filtrate,  as  it  comes  from  the  funnel,  in  a  test-tube  and  adding-  to 
this  a  little  HC1.  Not  the  slightest  cloudiness  should  result.  When 
this  point  is  reached  by  repeated  washing,  place  the  funnel  with  its 
contents  in  an  air-bath  at  about  100°  for  about  one  hour.  While  the 
precipitate  is  drying,  take  a  clean  porcelain  crucible,  ignite  it,  set 
aside  in  a  desiccator  to  cool,  and  when  cold,  weigh. 

The  precipitate  when  dry  is  transferred  as  carefully  as  possible 
to  the  weighed  porcelain  crucible.  The  filter  is  then  rolled  up  into  a 
small  cylinder,  a  platinum  wire  is  wrapped  around  it  and  it  is  then 
ignited  by  means  of  a  small  flame.  The  burning  filter  should  be  held 
over  the  crucible  which  should  be  placed  on  a  black  glazed  paper  so 
that  if  any  material  should  drop  down  it  would  not  be  lost  sight  of. 
The  flame  of  the  burner  should  be  directed  against  the  charred  filter 
paper  till  it  becomes  white  or  grayish.  This  ash  is  added  to  the  con- 
tents of  the  crucible.  A  drop  of  HN03  is  added  to  the  residue  in  the 
crucible  and  gently  warmed.  Then  a  drop  of  HC1  is  added  and  the 
whole  is  heated  on  a  water-bath  to  dryness.  The  crucible  is  now 
placed  on  a  clay  triangle  and  ignited  gently  till  the  residue  just 
begins  to  fuse  at  the  edges.  It  is  then  placed  in  a  desiccator  and 
weighed  when  cold. 

Example. — Total  urine  =  1450  c.c.     10  c.c.  are  taken  for  analysis: 

Crucible  +  AgCl  +  filter  ash  =  12.4554 
crucible  =  12.210 


AgCl  +  filter  ash  =      .2454 
filter  ash  =      .0001 


AgCl  =      .2453 

AgCl :  NaCl ::  weight  of  AgCl :  weight  of  NaCl. 
143.5  :  58.5  ::        0.2453  :    x 

x  =  0.1  g.  of  NaCl  in  10  c.c.  of  urine. 
10  :  0.1  ::  1450  :  y.         y  =  14.5  g.  of  NaCl  in  total  urine. 

For  gravimetric  work,  if  possible,  filter  paper  should  be  used 
that  has  been  washed  in  HC1  and  in  HF.  Such  paper  is  practically 
pure  cellulose  and  has  so  little  ash  that  this  can  be  disregarded.  If 
such  papers  cannot  be  obtained,  then  five  or  ten  ordinary  filter  papers 
should  be  ignited  as  just  given  and  the  ash  received  in  a  weighed 
crucible  and  weighed.  The  weight  of  ash  from  a  single  filter  paper 
can  then  be  ascertained. 


244  PHYSIOLOGICAL  CHEMISTRY. 


VI.     Determination  of  Total  Sulphuric  Acid. 

[a).     Volumetrically. 

Reagent. — Dissolve  30.5  g.  of  barium  chloride  (BaCl2  +  2  H20)  in 
water  and  dilute  to  one  liter.     Each  c.c.  =  10  mg\  of  S03. 

Execution. — To  50  or  100  c.c.  of  the  urine  add  5—10  c.c.  of  hydro- 
chloric acid  and  boil  for  %.  hour  in  order  to  break  up  conjugate 
sulphates.  Then  from  a  burette  run  in  the  barium  chloride  solution, 
1  c.c.  at  a  time,  as  long-  as  a  distinct  precipitate  continues  to  form; 
mix  and  allow  the  precipitate  to  subside  after  each  addition;  when 
the  formation  of  a  precipitate  becomes  indistinct  filter  off  after 
each  addition  a  few  c.c.  of  the  liquid  and  test  for  sulphuric  acid 
by  adding  a  few  drops  of  barium  chloride  solution  from  the  burette. 
If  a  precipitate  forms  pour  the  solution  back  into  the  beaker,  add 
more  barium  chloride,  mix  and  again  filter  off  a  small  portion.  Re- 
peat this  addition  of  reagent  and  testing  until  a  filtered  portion 
ceases  to  give  a  precipitate  with  barium  chloride.  Note  the  amount 
employed. 

The  contents  of  the  beaker  should  be  raised  to  boiling  after  each 
addition  of  barium  chloride. 

Suppose  17  c.c.  of  barium  chloride  does  not  precipitate  all  the 
sulphuric  acid  while  18  c.c'  does.  Take  the  same  amount  of  urine, 
treat  as  before,  then  run  in  the  full  amount  of  barium  chloride  solu- 
tion up  to  the  next  to  the  last  addition — that  is  17  c.c.  Mix  and  test 
a  little  of  the  filtered  solution;  now  add  the  reagent  in  smaller 
amounts  than  before — about  0.2  c.c.  at  a  time,  testing  after  each 
addition,  till  the  point  of  complete  precipitation  is  reached. 

Note  the  amount  of  barium  chloride  used.  Each  c.c.  =  10  mg. 
of  S03.     Calculate  the  amount  of  acid  present  in  the  24  hours'  urine. 

[b).  Gravimetrically. — Place  a  definite  amount  of  the  solution 
(50  or  100  c.c.  of  urine,  or  10  c.c.  of  the  unknown)  in  a  beaker,  dilute 
to  about  150  c.c.  and  acidify  with  a  few  drops  of  HC1.  Heat  the  solu- 
tion to  boiling  in  order  to  break  up  conjugate  sulphates.  Then  add 
ordinary  BaCl2  solution  very  slowly  and  with  constant  stirring  till  a 
slight  excess  has  been  added.  Boil  again  for  some  minutes  to  granu- 
late the  precipitate  and  then  set  aside  for  some  hours,  or  better  till 
next  day.  Filter  through  paper,  the  ash  of  which  is  known,  and  wash 
the  precipitate  with  hot  water  till  the  wash-water  ceases  to  give  a 
test  for  chlorides. 


QUANTITATIVE    ANALYSIS.  245 

Dry,  transfer  the  precipitate  to  a  weighed  crucible,  observing  the 
same  care  and  precaution  as  given  under  chlorides  (p.  243),  and  ignite. 
After  ignition  moisten  the  residue  in  the  crucible  with  a  drop  of  very 
dilute  H,S04;  place  the  crucible  on  a  clay  triangle  and  heat  with  a  very 
low  flame  till  the  fumes  of  S03  cease  to  be  given  off.  Then  ignite  the 
crucible  gently  and  place  in  a  desiccator  to  cool.  Weigh  the  crucible 
and  from  the  weight  obtained,  deduct  that  of  the  crucible  and  of  the 
filter  ash;  the  difference  is  the  weight  of  the  BaS04.  Now  calculate 
the  amount  of  SOs  present  in  the  24  hours'  urine. 

(c).    *8eparate  determination  of  simple  and  conjugate  sulphates.— The 

methods  given  under  a  and  b  yield  the  total  amount  of  S03  present  in 
the  urine.  To  ascertain  the  amount  of  S03  present  as  simple  sul- 
phate and  as  conjugate  sulphate  proceed  as  follows: 

Acidulate  50  or  100  c.c.  of  urine  with  acetic  acid,  add  BaCl2  in 
excess  and  warm  on  the  water-bath  for  y2 — 1  hour.  Then  filter 
through  a  filter  of  known  ash,  wash,  dry  and  ignite  as  above.  This 
gives  the  amount  of  S03  present  as  simple  sulphate. 

The  filtrate  from  the  BaS04  precipitate  contains  the  conjugate 
sulphates.  To  break  these  up,  acidulate  strongly  with  HC1  and  boil 
for  some  time:  add  more  BaCl2  if  necessary,  allow  the  precipitate  of 
BaS04  to  settle,  then  transfer  to  a  filter,  wash,  dry,  and  ignite  as 
above.  Calculate  the  amount  of  S03  present  as  conjugate  sulphates. 
Also  determine  the  ratio  existing  between  this  and  the  amount  of 
S03  in  the  simple  sulphates. 

VII.     Determination  of   Phosphoric  Acid. 

a  .     VolumetricaUy. 

Beagent8.—l.—UroMiv/m  aeetatt  solution. — Dissolve  35  g.  of  crystal- 
lized uranium  acetate  in  a  liter  of  water.  This  solution  must  be 
standardized  against  a  standard  solution  of  sodium  phosphate  so  that 
each  c.c.  =  5  mg.  of  P205. 

For  this  purpose  dissolve  10.0845  g.  of  crystallized  Na2HP04  + 
12  HjO  in  water  and  make  up  to  1  liter;  each  c.c.  =  2  mg.  of  P2Os; 
50  c.c.  =  0.1  g.  of  P205.  20  c.c.  of  the  uranium  acetate  solution  should 
equal  50  c.c.  of  this  phosphate  solution.  If,  on  titration  this  is  not 
found  to  be  the  case,  calculate  and  make  the  necessary  dilution.  The 
titration  is  done  according  to  the  directions  given  below. 

Sodium  phosphate  effloresces  very  rapidly.  The  pure  crystals 
should  be  selected  and  weighed  as  rapidly  as  possible,  best  in  a  weigh- 
ing-bottle. 


246  PHYSIOLOGICAL  CHEMISTRY. 

2. — 3  per  cent,  acetic  acid  solution. — If  uranium  nitrate  is  used 
this  solution  must  contain  in  addition  10  per  cent,  of  sodium  acetate 
in  order  to  combine  with  the  nitric  acid  which  would  be  set  free  in 
the  reaction. 

3. — Tincture  of  cochineal,  or  a  solution  of  potassium  ferrocyanide 
to  serve  as  indicator. 

Execution.  —Place  50  c.c.  of  urine,  or  10  c.c.  of  the  unknown 
diluted  to  50 — 100  c.c,  in  a  beaker  or,  better  still,  in  a  small  Erlen- 
meyer  flask;  add  5  c.c.  of  the  acetic  acid  solution,  a  few  drops  of  the 
cochineal  tincture,  and  heat  to  boiling.  Now  run  in  from  a  burette 
the  uranium  acetate  solution,  stirring-  well,  till  the  liquid  becomes 
faintly  but  distinctly  green  and  remains  so  when  heated  again  to  the 
boiling  point. 

Note  the  number  of  c.c.  employed;  each  c.c.  =  5  mg.  of  P2Os. 
Calculate  the  amount  present  in  the  24  hours'  urine. 

Potassium  ferrocyanide  solution  can  be  used  as  an  indicator 
instead  of  cochineal.  For  this  purpose  place  a  series  of  drops  of  the 
solution  on  a  porcelain  surface,  or  on  a  filter  paper,  and  after  each 
addition  of  uranium  acetate,  remove  a  drop  from  the  beaker,  by 
means  a  glass  rod,  and  add  it  to  the  ferrocyanide;  a  reddish-brown 
color  indicates  the  end-reaction, — that  uranium  acetate  has  been 
added  in  excess. 

(b).  Gravimetrically. — Phosphoric  acid  cannot  be  estimated  grav- 
imetrically  in  solutions  containing  calcium,  iron  or  other  metals  pre- 
cipitable  in  alkaline  solution  by  this  acid,  unless  it  is  first  of  all 
separated  from  these  metals  by  means  of  ammonium  molybdate. 
This  step  would  be  necessary,  therefore,  in  order  to  determine  the 
phosphoric  acid  in  urine  gravimetrically.  In  the  absence  of  such 
interfering  metals,  as  in  the  case  of  the  unknown  given,  the  phos- 
phoric acid  can  be  determined  directly  according  to  the  following 
method: 

Place  10  c.c.  of  the  solution  in  a (  small  beaker,  dilute  to  about 
50  c.c.  and  render  slightly  alkaline  with  ammonium  hydrate.  Then 
add  magnesia  mixture,  drop  by  drop,  till  no  further  precipitation 
results.  While  adding  this  reagent  the  liquid  should  be  stirred  con- 
stantly and  vigorously,  taking  care,  however,  that  the  rod  does  not 
touch  the  sides  of  the  beaker.  Wherever  the  rod  touches  the  beaker 
the  precipitate  adheres  tenaciously.  Allow  the  liquid  to  stand  about 
a  quarter  of  an  hour,  then  add  about  one-third  volume  of  NH4OH, 
mix  and  set  aside  for  a  couple  of  hours,  or  over  night.     Filter  through 


QUANTITATIVE  ANALYSIS.  247 

a  paper  of  known  ash  and  wash  with  dilute  ammonia  (1  :  4)  till  the 
wash-water  is  free  from  chlorides.  Before  testing  the  wash-water 
with  silver  nitrate  render  it  slightly  acid  with  HN03.  Dry,  transfer 
the  precipitate  to  a  weighed  crucible  and  ignite  the  filter  according 
to  the  directions  given  under  chlorides  (p.  243).  Place  the  crucible 
on  a  triangle,  apply  a  small  flame  and  gradually  increase;  finally 
ignite  for  about  ten  minutes  over  a  Detroit  burner  or  blast-lamp. 
The  residue  should  be  white  or  nearly  so.  If  it  is  dark,  cool  the  cru- 
cible, add  one  drop  of  cone.  HXOa  and  then  place  on  a  water-bath  to 
drive  off  the  acid:  finally  ignite  again  for  a  few  minutes.  Cool  in  the 
desiccator  and  weigh.  Deduct  the  weight  of  the  crucible  and  of 
the  filter-ash  from  the  weight  obtained.  This  gives  the  weight  of 
Mg2P,07.    Calculate  the  amount  of  P205  present. 

In  this  method  the  phosphoric  acid  is  precipitated  as  magnesium 
ammonium  phosphate  (triple  phosphate).  On  ignition  this  is  con- 
verted into  magnesium  pyrophosphate  (Mg2P207): 

Xa2HP04  +  MgCl2  4-  XH,OH  =  XH4MgPO,  +  2  XaCl  +  H20. 
2  XH.MgPO,  -  ignition  =  Mg2Ps07  +  2  XH3  +  H20. 

VIII.     Determination  of  Glucose. 

(a).     Volvmetrically. — By  titration  with  Fehling's  solution. 

Rftiij,  ,,i. — Dissolve  34.64  g.  of  pure,  crystallized  copper  sulphate  in 
water  and  make  up  to  %  liter.  Likewise  dissolve  173  g.  of  Rochelle 
salts  (potassium  sodium  tartrate)  and  60  g.  of  sodium  hydrate  each  in 
200  c.c.  of  water.     Combine  the  two  solutions  and  make  up  to  yi.  liter. 

The  two  solutions  thus  prepared  are  usually  united  and  consti- 
tute then  the  so-called  Fehling's  solution.  Inasmuch  as  Fehling's 
solution  deteriorates  on  keeping,  it  is  preferable  to  keep  the  two 
constituents  separate,  mixing  them  in  equal  portions  just  before  use. 

10  c.c.  of  Fehling's  solution  =  50  mg.  of  glucose. 

ution. — Preliminary  trial. — Run  10  c.c.  of  Fehling's  solution 
from  a  pipette  into  a  200  c.c.  Erlenmeyer  flask  (or  porcelain  evaporat- 
ing dish),  add  about  40  c.c.  of  water  and  heat  to  boiling;  now  run  in 
from  a  burette  the  urine  or  sugar  solution  1  or  2  c.c.  at  a  time  till  the 
blue  color  has  disappeared  and  a  faint  yellow  color  remains.  This 
can  best  be  seen  by  holding  the  flask  in  an  inclined  position  before 
a  window.     Allow  the  precipitate  to  partly  subside  after  each  addi- 


248  PHYSIOLOGICAL  CHEMISTRY. 

tion,  the  better  to  see  the  color.  "When  decoloration  takes  place  note 
the  amount  of  urine  or  solution  used  and  calculate  the  amount  of 
sugar  present  in  100  ex. 

This  preliminary  trial  will  give  approximately  the  strength  of 
the  sugar  solution  used.  The  above  factor  for  Fehling's  solution 
(10  c.c.  =  50  mg.  of  glucose)  holds  true  only  for- solutions  of  a  certain 
strength,  namely  about  0.5  per  cent,  glucose.  Having  ascertained 
the  approximate  strength  of  the  sugar  solution  dilute  this  so  that 
it  will  contain  about  0.5  per  cent,  of  sugar.  Note  carefully  the 
quantity  of  the  sugar  solution  taken  and  the  volume  to  which  it 
is  diluted. 

Final  determination. — Calculate  approximately  the  number  of  c.c. 
of  the  diluted  sugar  solution  necessary  to  reduce  10  c.c.  of  Fehling's 
solution.  Now  measure  out  10  c.c.  of  Fehling's  solution,  dilute  and 
heat  as  above.  Then  run  in  nearly  the  calculated  amount  of  diluted 
sugar  solution,  heat  to  boiling  and  examine  for  the  end  reaction 
according  to  the  directions  given  above.  If  the  solution  contains 
unreduced  copper,  that  is,  is  colored  blue,  add  )4  c.c.  of  the  sugar 
solution,  raise  to  boiling  and  examine  as  before.  Continue  the  addi- 
tion of  the  sugar  solution,  in  portions  of  y2  c.c.  till  the  blue  color  just 
disappears.  In  case  of  doubt  filter  some  of  the  liquid  into  a  test-tube. 
If  this  is  nearly  full  and  held  against  a  white  surface  a  trace  of  blue 
color  can  be  readily  detected.  Another  procedure  is  to  acidulate  a 
few  c.c.  of  the  filtrate  with  acetic  acid  and  then  add  a  drop  or  two  of 
potassium  ferrocyanide.  If  copper  is  present  a  brown  precipitate  or 
coloration  will  result. 

Example. — On  preliminary  trial  10  c.c.  of  Fehling's  solution 
required  between  5  and  6  c.c.  of  the  urine.  This  represents  0.8  to  1.0 
g.  of  sugar  in  100  c.c.  The  original  solution  is  therefore  about  twice 
as  strong  as  it  should  be.  Consequently  50  c.c.  of  this  solution  were 
diluted  to  100  c.c. 

On  repeating  the  titration  10  c.c.  of  Fehling's  solution  required 
10.3  c.c.  of  the  diluted  urine.  This  amount,  therefore,  contains  50 
mg.  of  glucose.  The  amount  of  sugar  present  in  the  entire  solution 
is  then  ascertained  by  the  proportion:  10.3  :  50  ::  100  :  x.  x  —  485  mg. 
=  0.485  g.  of  glucose  in  100  c.c.  of  the  diluted  or  in  50  c.c.  of  the  undi- 
luted urine.  If  now  the  total  24  hours'  urine  is  4500  c.c.  the  amount 
of  sugar  present  will  be  given  by  the  proportion: 

50  :  0.485  ::  4500  :  x.        x  -  43.65  g.  of  glucose. 


QUANTITATIVE   ANALYSIS.  249 

If  albumin  is  present  this  should  be  removed,  since  it  combines 
with  copper  and  also  interferes  with  the  settling  of  the  precipitate. 
This  can  best  be  done  as  follows:  To  100  c.c.  of  the  urine  add  10—15 
c.c.  of  saturated  salt  solution,  acidulate  distinctly  with  a  few  drops 
of  acetic  acid  and  boil  several  minutes,  then  cool,  make  up  to  100  c.c. 
and  filter  through  a  dry  filter. 

Inasmuch  as  sugar  solutions  are  prone  to  fermentation  the  quan- 
titative examination  should  be  made  as  early  as  possible. 

(&)*.     By  fermentation. 

1.— Prom  the  difference  in  specific  gravity,  before  and 
after  fermentation.  Determine  the  specific  gravity  of  the 
fresh  urine  and  reduce  this  to  15°.  Take  200  c.c,  add 
about  1  gram  of  yeast  and  set  aside  in  a  bottle  closed  with 
a  perforated  stopper  for  24—48  hours  at  a  temperature  of 
25°— 30°  C.  Then  filter  off  a  small  portion  and  test  with 
Pehling's  solution.  If  reduction  takes  place  continue  the 
fermentation  till  all  the  sugar  has  disappeared.  ^J.Then 
filter  the  urine,  determine  the  specific  gravity  and  reduce 
to  15°.  The  difference  between  the  two,  multiplied  by  230 
(Roberts'  factor),  gives  the  amount  of  sugar  in  grams  in 
100  c.c.     Calculate  the  amount  in  the  24  hours'  urine. 

The  results  obtained  by  this  method  are  quite  satisfac- 
tory, especially  if  the  urinometer  reads  to  four  decimal 
places,  or  the  specific  gravity  is  determined  by  the  pic- 
nometer. 

The  above  factor  holds  true  in  the  case  of  urines  con- 
taining a  small  amount  of  sugar.  If  the  specific  gravity  is 
high  it  is  advisable  to  dilute  the  urine  as  given  on  p.  250. 

2. — *From  the  amount  of  carbonic  acid  formed. 

Einhorn's  saccharimeter  and  similarly  constructed  in- 
struments can  be  used. 

Sugar  on  fermentation  with  yeast  breaks  up  into  alco- 
hol and  carbonic  acid: 

C6HI206  =  2  C,H60  +  2  C02. 


250  PHYSIOLOGICAL  CHEMISTRY. 

If  the  fermentation  takes  place  in  a  U-shaped  tube,  one 
arm  of  which  is  closed  and  graduated  the  carbonic  acid 
given  off  will  accumulate  in  the  closed  arm  and  the  volume 
of  gas,  or  the  percentage  of  sugar  that  this  corresponds  to, 
can  be  read  off  directly.  For  Einhorn's  instrument  10  c.c. 
of  urine  are  taken,  1  g.  of  yeast  added,  mixed  thoroughly 
and  the  mixture  is  filled  into  the  fermenting  tube.  This  is 
set  aside  for  15 — 20  hours.  The  volume  of  gas  gives  directly 
the  percentage  of  sugar  present.  If  the  urine  is  rich  in 
sugar,  that  is,  possesses  a  high  specific  gravity,  it  should 
be  diluted,  as  follows: 

Specific  gravity  1.018—1.024,  with.  2  volumes  of  water. 
1.024—1.028,     "      5        "         " 
1.028—1.038,     "10 

Even  with   this   precaution   the   results   are   far  from 
satisfactory.     The  rate  of  fermentation  is  influenced  by  the 
temperature.     The  volume  of  the  gas  is  affected  by  temper- 
ature  and   barometric   pressure.     The   method,  therefore, ' 
cannot  be  recommended. 

3. — Prom  the  amount  of  alcohol  formed. 

(c)*.  Polarization  method. — This  gives  reliable  results, 
though  a  trifle  low.  The  expense  of  the  instrument  is  such 
as  to  preclude  its  general  use  (see  p.  35). 

(cZ)*.  Ch"avimetrically. — The  amount  of  sugar  present  in 
a  solution  maybe  ascertained  by  collecting  the  Cu20  formed 
on  heating  with  Pehling's  solution  and  converting  this  into 
metallic  copper  by  heating  it  in  a  current  of  hydrogen. 
Allihn's  tables  give  the  amount  of  glucose  corresponding 
to  the  weight  of  copper  found. 


QUANTITATIVE   ANALYSIS.  251 


IX.     Determination  of  Urea. 


1. — Liebig's  Method. — This  is  more  nearly  an  estimate  of  the  total 
nitrogen  present  in  the  urine. 

Reagents.— 1. — Mercuric  nitrate  solution. — Dissolve  77.2  g.  of  mer- 
curic oxide  (or  71.48  g.  of  mercury)  in  nitric  acid  and  evaporate  to 
drive  off  the  excess  of  acid,  then  add  water  gradually,  stirring  well, 
and  dilute  to  one  liter.  Each  c.c.  should  equal  10  mg.  of  urea.  The 
solution  should  be  standardized  against  a  2  per  cent,  urea  solution. 

2. — Baryta  mixture. — Combine  one  volume  of  cold  saturated  bar- 
ium nitrate  with  two  volumes  of  cold  saturated  barium  hydrate.  This 
is  used  to  remove  phosphoric  acid. 

3. — Solution  of  sodium  carbonate  containing  53  g.  of  the  salt  in 
one  liter  of  water.     This  serves  to  indicate  the  end  reaction. 

Execution. — To  40  c.c.  of  the  urine  add  20  c.c.  of  baryta  mixture, 
mix  well  and  filter  through  a  dry  filter.  Place  15  c.c.  of  the  clear 
filtrate,  which  corresponds  to  10  c.c.  of  the  original  urine,  in  a  small 
beaker  and  run  in  the  mercuric  nitrate  solution  from  a  burette,  1  c.c. 
at  a  time,  stirring  well  and  testing  with  sodium  carbonate  after  each 
addition.  This  is  best  done  by  placing  a  series  of  drops  of  sodium 
carbonate  by  means  of  a  glass  rod  on  a  flat  porcelain  surface,  and 
after  each  addition  of  reagent  adding  a  drop  of  the  mixture  from  the 
beaker  to  the  drop  of  sodium  carbonate.  Continue  the  addition  of 
the  mercury  solution  till  a  distinct  yellow  coloration  is  produced  by 
the  sodium  carbonate.  It  is  well  to  wait  a  minute  for  the  develop- 
ment of  the  color  before  adding  more  reagent. 

Now  repeat  the  titration  with  a  new  portion,  running  in  the  full 
amount  up  to  the  point  short  of  the  yellow  color  and  from  this  on  add 
0.2  c.c.  at  a  time  till  the  end  reaction  appears.  Note  the  number  of 
c.c.  required:  each  c.c.  =  10  mg.  of  urea.  Calculate  the  amount  in 
the  24  hours'  urine. 

If  an  aqueous  "unknown"  of  urea  is  to  be  titrated,  take  10  c.c. 
and  add  the  mercuric  solution  to  this.  The  baryta  mixture  should 
not  be  added,  when  no  phosphates  or  sulphates  are  present. 

If  much  albumin  is  present  it  should  be  removed  by  the  aid  of 
heat  and  acetic  acid.     The  NaCl  method  is  not  applicable. 


252  PHYSIOLOGICAL  CHEMISTRY. 

The  above  is  the  method  as  ordinarily  and  most  easily  employed. 
More  exact  results  can  be  obtained  if  the  chlorides  present  are 
removed  by  precipitation  with  just  sufficient  silver  nitrate.  Why  is 
this  desirable? 

Again,  in  the  above  method  as  seen  from  the  following  equation, 
nitric  acid  is  set  free. 

2  CO(NH2)2  +  4  Hg(N03)2  +  3  H20  = 

2  CO(NH2)2 .  Hg(N03)2 .  3  HgO  +  6  HNOs. 

This  free  acid  tends  to  alter  the  composition  of  the  precipitate 
and  to  dissolve  it,  hence  the  solution  should  be  carefully  neutralized. 

2. — * 'Hufner 's  method. — This  depends  upon  tne  fact  that 
urea  is  decomposed  by  an  alkaline  solution  of  sodium 
hypobromite  into  nitrogen,  carbonic  acid  and  water. 

Reagent. — Alkaline  hypobromite  solution.  Dissolve  100 
g.  of  sodium  hydrate  in  250  c.c.  of  water  and  when  cold 
add  slowly  and  cautiously  25  c.c.  of  bromine.  Keep  in  a 
cool,  dark  place. 

The  above  quantity  of  reagent  is  necessary  when  using 
Hiifner's  apparatus,  but  when  the  small  modified  form,  as 
that  of  Doremns,  is  used  it  is  unnecessarily  large.  In  that 
case  it  is  better  to  add  1  c.c.  of  bromine  to  10  c.c.  of  the 
NaOH  solution  just  before  use. 

Execution. — Place  1  c.c.  of  the  urine  in  the  lower  bulb 
of  Hiifner's  apparatus,  then  fill  it  full  of  water  up  to  the 
farther  end  of  the  perforation  in  the  stopper.  Now  close 
the  stopper.  Fill  the  remainder  of  the  apparatus  and  the 
measuring  tube  with  the  reagent.  Close  the  mouth  of  the 
tube  with  a  finger  and  invert  over  the  apparatus  so  that 
no  air  enters,  and  place  it  into  position.  When  this  is 
done  open  the  stop-cock;  when  the  reagent  passes  into  the 
bulb  where  the  urine  is  and  sets  nitrogen  free.  This  col- 
lects above  in  the  measuring  tube.  In  about  a  half  hour 
the  operation  is  completed  and  the  volume  of  gas  can  be 
measured,  using  the  proper  precautions. 


QUANTITATIVE   ANALYSIS.  253 

From  the  volume  of    nitrogen  the  weight  of  the  urea 
can  be  calculated  according  to  the  formula: 

_  _  v  (b  —  b') 


354.5  +  760  (1  +  0.003665  t) 


G  =  weight  of  urea;  v  =  volume  of  gas;  b  =  baromet- 
ric pressure;  b'  aqueous  tension  at  t°;  t  =  temperature. 

The  above  procedure  is  too  expensive  for  ordinary  clinical  work. 
The  principle,  however,  can  be  utilized  advantageously  in  the  small 
modified  apparatus,  or  ureometer  of  Doremus.  The  results,  how- 
ever, are  usually  a  trifle  low.  The  instrument  is  filled  to  the  bend 
with  a  mixture  cf  equal  parts  of  hypobromite  and  water.  1  c.c.  of 
the  urine  is  then  introduced  by  means  of  a  nipple  pipette.  The  vol- 
ume of  nitrogen  given  off  gives  directly  the  amount  of  urea  in  1  c.c. 
of  urine. 

3. — Other  methods  for  determination  of  urea  are  based 
upon  its  decomposition  into  ammonia  and  carbonic  acid, 
but  for  ordinary  purposes  they  are  not  applicable. 

The  same  is  true  of  Morner  and  Sjoqvist's  method,  in 
which  all  the  nitrogen  constituents  of  urine,  except  urea, 
are  precipitated  by  addition  of  BaCl2,  baryta  mixture, 
alcohol  and  ether.  The  filtrate  is  concentrated  and  in  it 
the  nitrogen  is  determined  by  the  Kjeldahl  process. 

4. — The  specific  gravity  affords  an  approximate  idea  of 
the  amount  of  urea  present,  provided  sugar  and  albumin 
are  absent  and  the  volume  of  urine  and  the  amount  of 
chlorides  are  about  normal. 

A  specific  gravity  of  1.010  indicates  about  1.0  per  cent,  of  urea; 
1.015  indicates  about  1.5  per  cent.;  and  1.020  indicates  a  little  less  than 
2  per  cent,  of  urea. 

Above  this  point  the  variation  is  too  great  to  be  of 
any  value. 


254 


PHYSIOLOGICAL  CHEMISTRY. 


X.     Determination  of   Uric  Acid. 


1. — Method  of  Heintz. — Place  200  c.c.  of  the  filtered  urine  in  a 
"beaker,  add  5  c.c.  of  cone,  hydrochloric  acid,  mix,  cover  and  set  aside 
for  24 — 48  hours  in  a  cool  place.  Filter  througii  a  previously  dried 
and  weighed  filter,  transfer  the  crystals  to  the  filter,  wash  with 
small  portions  of  cold  water  till  the  chlorides  are  removed,  then 
dry  at  100—110°  and  weigh.  The  difference 
in  the  two  weighings  represents  the  uric  acid. 
But  as  uric  acid  is  not  wholly  insoluble  in 
water,  some  of  it  is  in  solution.  Therefore, 
measure  the  filtrate  and  wash-water  and  for 
every  100  c.c.  add  0.0048  g.  to  the  amount  of 
uric  acid  as  found  above.  Now  calculate  the 
total  amount  in  the  24  hours'  urine.  By  mak- 
ing this  correction  the  method  is  as  accurate 
as  the  following  one.  If  urates  are  present 
they  should  be  brought  into  solution  by  the  aid 
of  heat  or  by  the  addition  of  sodium  hydrate. 
Albumin  must  be  removed. 

Filter  paper  should  not  be  weighed  in  the 
open  air  but  should  be  placed  in  a  weighing 
bottle  or  well  corked  test-tube. 

Instead  of  using  a  filter  paper  for  the  above 

or  similar  purposes  it  is  preferable  to  use  an 

asbestos  filter  in  connection  with  a  Chapman 

The  filter  is  shown  in  the  accompanying  figure  (Fig.  5). 


2. — Method  of  Ludwig. 

Reagents. — 1. — Ammoniacal  silver  nitrate  solution.  Dissolve  26  g. 
of  silver  nitrate  in  water,  add  ammonium  hydrate  till  the  precipitate 
which  first  forms  redissolves,  then  dilute  to  100  c.c. 

2. — Magnesia  mixture. — Dissolve  10  g.  of  magnesium  chloride  in 
water,  add  ammonium  hydrate  in  strong  excess  and  then  ammonium 
chloride  till  the  precipitate  is  redissolved.  Dilute  to  100  c.c.  The 
solution  should  be  strongly  ammoniacal. 


3. — Sodium  or  potassium  sulphide  solution. — Dissolve  15  g.  of  potas- 
sium hydrate  (or  10  g.  of  sodium  hydrate),  free  from  nitrous  and 
nitric   acids,  in  100  c.c.  of  water.     Saturate   one-half  the  solution 


QUANTITATIVE   ANALYSIS.  255 

with  hydrogen  sulphide  which  forms  KHS.     Then  combine  the  two 
solutions  forming-  K2S. 

Execution. — TolOOc.c.  of  urine  add  a  mixture  of  lOc.c.  of  the  silver 
solution  and  10  c.c.  of  magnesia  mixture  and  mix.  If  when  making 
this  mixture  silver  chloride  is  thrown  down  more  ammonia  is  to  be 
added;  if  magnesium  hydrate  precipitates  more  ammonium  chloride 
is  needed.  Allow  the  precipitate  to  settle,  then  filter  and  wash  2 — 3 
times  with  water  to  which  a  little  ammonia  has  been  added.  When 
the  liquid  has  drained,  transfer  by  means  of  a  glass  rod  the  moist 
precipitate  to  the  beaker  in  which  the  precipitation  was  made,  place 
the  beaker  under  the  funnel  and  wash  the  residue  on  the  filter  with  a 
boiling  mixture  of  10  c.c.  of  potassium  sulphide  solution  and  10  c.c.  of 
water.  Heat  on  a  water-bath  for  some  time  and  when  the  entire 
precipitate  is  black,  filter  through  the  filter  previously  used.  Collect 
the  filtrate  in  a  small  dish  and  wash  the  filter  with  hot  water.  To 
the  combined  filtrate  and  wash-water  add  5  c.c.  of  dilute  hydrochloric 
acid  and  concentrate  to  10 — 15  c.c.  Set  aside  to  cool:  in  about 
half  an  hour  all  the  uric  acid  will  have  crystallized  out.  Filter 
through  a  glass-wool  or  asbestos  filter,  or  through  paper  previously 
dried  at  110  and  weighed.  Wash  with  a  little  water,  then  dry  and 
wash  three  times  with  carbon  bisulphide  to  remove  sulphur,  then  with 
ether.  ^Finally  dry  at  110'  and  weigh.  The  difference  between  the 
two  weighings  gives  the  amount  of  uric  acid.  Calculate  the  amount 
present  in  the  24  hours"  urine. 

If  albumin  is  present  it  should  be  removed  according  to  the 
method  on  p.  249. 

3. — *Haycraff8  method. — This  is  a  volumetric  method 
based  upon  the  same  principle  as  the  preceding-  and  gives 
somewhat  higher  results.  The  uric  acid  is  thrown  out  of 
solution  and  the  precipitate  is  washed  as  above.  Then  it 
is  dissolved  in  nitric  acid  and  the  dissolved  silver  titrated 
with  a  ft  potassium  sulphocyanate  solution  (see  p.  241). 
Each  c.c.  used  =  3.36  mg.  of  uric  acid. 

4. — *Czapek'8  method. — This  method  is  the  complement  of 
Haycraft's.  That  is,  instead  of  estimating  the  silver  in  the 
precipitate,  the  silver  in  the  nitrate  is  determined  and  the 
difference  between  this  amount  and  the  total  amount  added 


256  PHYSIOLOGICAL  CHEMISTRY. 

gives  the  amount  of  silver  that  combined  with  uric  acid. 
The  results  are  likewise  higher  than  by  Ludwig's  method. 

5. — Hopkins1  method. — This  depends  upon  the  fact  that  uric  acid  is 
completely  precipitated  from  urine  on  saturation  with  NH4C1  as 
ammonium  urate.  Furthermore,  uric  acid  can  be  titrated  in  warm, 
H2S04  solution  with  KMn04.  Instead  of  NH4C1  other  ammonium  salts 
can  be  employed  and  the  addition  of  10  per  cent,  of  these  salts  is 
sufficient.     The  method  as  modified  by  Folin  is  as  follows: 

To  100  c.c.  of  the  urine  add  10  g.  of  (NH4)2S04,  stir  till  dissolved, 
then  add  NH4OH  to  slight  but  distinct  alkaline  reaction.  The 
ammonium  urate  is  allowed  to  settle  for  two  hours.  The  solution  is 
filtered  and  the  precipitate  washed  with  a  10  per  cent.  (NH4)2S04 
solution,  till  free  from  chlorides.  It  is  then  rinsed  into  a  small 
beaker,  diluted  to  100  c.c.  and  15  c.c.  of  concentrated  H2S04  added. 
Heat  to  60°,  then  titrate  immediately  with  ^  KMn04,  Each  c.c.  of 
the  KMn04  solution  corresponds  to  3.75  mg.  of  uric  acid.  A  correc- 
tion of  1  mg.  is  to  be  added  to  the  end  result. 

The  method  is  rapid,  easy  of  execution  and  gives  almost  as  good 
results  as  that  of  Ludwig.  The  KMn04  solution  can  be  prepared  by 
dissolving  1.578  g.  of  the  pure  crystals  in  water  and  diluting  to  one 
liter.  It  should  then  be  tested  against  &  oxalic  acid.  Ten  c.c.  of  the 
latter  are  placed  in  a  beaker,  diluted  with  a  little  water,  H2S04  added 
and  the  mixture  warmed  to  60°.  The  KMn04  solution  is  then  run  in  to 
a  permanent  pink  color.  Twenty  c.c.  of  the  latter  should  be  con- 
sumed; if  not,  calculate  the  factor.     Thus,  10  c.c.  of  ^  (—  20  c.c.  ^) 

20 
oxalic  acid  required  19.6  c.c.  of  the  KMn04;  ^-^  =   1.0204,   the  factor 

of  the  KMn04  solution.  That  is  to  say.  the  number  of  c.c.  of  this 
KMn04  used  multiplied  by  this  factor  gives  the  number  of  c.c.  of  ^ 
KMn04  that  this  corresponds  to. 


XI.     Alloxuric  Bodies  and  Bases. 

Uric  acid  and  the  nuclein  bases  are  known  to  possess  an 
alloxan  and  an  urea  group  in  their  molecule.  Moreover 
they  are  all  precipitated  on  boiling  with  a  solution  of  copper 
sulphate  and  a  reducing  agent  (see  p.  144,  Exp.  4).  The 
term  alloxuric  bodies  has  been  given  to  all  those  consti- 


QUANTITATIVE  ANALYSIS.  257 

tuents  of  urine  which  contain  the  two  groups  mentioned. 
Deducting  from  the  alloxuric  bodies  the  uric  acid  present 
in  the  urine  leaves  the  alloxuric  bases.  The  latter,  there- 
fore, include  the  nuclein  bases,  as  well  as  other  related  com- 
pounds which  have  not  as  yet  been  isolated  from  the  urine. 
The  following  members  of  the  xanthin,  or  nuclein  group 
may  be  obtained  from  urine  (see  p.  153):  Xanthin,  hypo- 
xanthm,  guanin,  paraxanthin,  heteroxanthin,  methyl  xan- 
thin, carnin,  episarkin  and  adenin.  They  are  ordinarily 
present  in  very  small  amount  but  may  be  appreciably 
increased  in  disease,  as  in  leukaemia.  The  separation  of 
these  bases  is  a  long  and  tedious  process.  For  the  details 
of  isolation  see  Vaughan  and  Novy — Ptomains  and  Leuco- 
mains. 

ESTIMATION  OF  ALLOXURIC  BODIES  AND  BASES. 

The  reagents  employed  are  a  13  per  cent,  solution  of  copper  sul- 
phate: a  solution  of  sodium  acid  sulphite  (1  :  2);  and  a  10  per  cent, 
solution  of  barium  chloride.     The  method  is  as  follows: 

Place  100  c.c.  of  the  albumin  free  urine  in  a  beaker  and  boil, 
then  add  10  c.c.  of  the  copper  sulphate  solution  and  10  c.c.  of  the 
sodium  acid  sulphite  solution  and  boil  a  few  minutes.  Then  add  5  c.c. 
of  the  barium  chloride  solution  in  order  to  cause  the  precipitate  to 
settle  more  readily.  Let  stand  two  hours.  Then  transfer  to  a  small 
plaited  filter  and  wash  five  times  with  water  heated  to  60°.  Now 
place  the  filter  and  contents  in  a  Kjeldahl  flask,  and  determine  the 
nitrogen  present  according  to  the  Kjeldahl  method  (p.  258). 

A  blank  experiment  must  be  made,  using  a  clean  filter  paper, 
instead  of  the  one  with  the  precipitate.  The  number  of  c.c.  of  deci- 
normal  ammonia  which  is  found  in  this  blank  experiment  must  be 
deducted  from  the  number  of  c.c.  found  above.  The  difference  repre- 
sents the  number  of  c.c.  of  deci-normal  ammonia  formed  from  the 
alloxuric  bodies  in  100  c.c.  of  the  urine.  Therefore,  this  number 
multiplied  by  the  deci-normal  factor  of  nitrogen  gives  the  nitro- 
gen in  the  alloxuric  bodies. 

In  another  portion  of  the  urine  determine  the  amount  of  the  uric 
acid  according  to  the  Salkowski-Ludwig  method  as  given  on  p.  255. 
Calculate  the  amount  of  uric  acid  contained  in  100  c.c.  of  the 
urine,  and  then  the  nitrogen  contained  in  this  amount  of  uric  acid. 


258 


PHYSIOLOGICAL   CHEMISTRY. 


Subtracting'  the  nitrogen  of  uric  acid  from  the  nitrogen  of 
alloxuric  bodies  gives  the  nitrogen  of  alloxuric  bases  in  100  c.c. 

The  ratio  of  the  nitrogen  of  alloxuric  bases  to  the  nitrogen  of 
uric  acid  is  about  1  to  4  in  normal  urine.  In  leukaemia  it  may  be 
Itol. 

The  class  is  divided  into  sets  of  three.  One  student  determines 
the  alloxuric  bodies;  another  the  uric  acid  according  to  Ludwig,  and 
the  third  determines  total  nitrogen.  These  three  determinations  are 
made  with  the  same  urine. 

XII.     Determination  of   Total  Nitrogen. 

1. — KjeldahVs  method. — This  method  is  based  upon  the  fact  that 
nitrogenous  substances  on  prolonged  heating  with  H2S04  are  com- 
pletely oxidized  and  the  nitrogen  present  is  converted  into  ammonia. 


Fig.  6. 
The  ammonium  sulphate  is  then  decomposed  by  distillation  with  an 
alkali  and  the   ammonia  that  distils  over  is  collected  in  a  known 
quantity  of  an  ^  acid.     On  determining  by  titration  with  ^  alkali 


QUANTITATIVE   ANALYSIS. 


259 


the  amount  of  acid  left  unneutralized,  the  difference  between  this 
and  the  amount  taken  represents  the  number  of  c.c.  of  T\  NH3  dis- 
tilled: multiplying  this  by  the  f%  factor  of  N,  the  amount  of  N  present 
is  at  once  obtained. 

The  method  is  carried  out  as  follows:  Place  5  c.c.  of  urine  in  a  250 
c.c.  Kjeldahl  flask.  (Fig.  6).  Add  15  c.c.  of  H^SO*.  and  %  g.  of  powdered 
CuS04:  heat  on  a  wire  gauze  under  the  hood  till  foaming  ceases, 
then  add  10  g.  of  powdered  K2S04  and  continue  gently  boiling  till  the- 


liquid  is  light  green.  Finally  add  a  little  powdered  KMn04,  on  the 
point  of  a  knife,  in  order  to  complete  the  oxidation,  and  heat  till  the 
liquid  is  light  green  in  color.  Allow  to  cool,  then  transfer  the  con- 
tents to  a  liter  Erlenmeyer  flask.  Rinse  out  the  digestion  flask 
several  times  with  water  and  add  this  to  the  acid  solution.  Dilute 
the  contents  of  the  flask  to  about  500  c.c.  and  cool.  Add  a  little  pow- 
dered talc  on  the  end  of  a  knife.  Insert  in  the  neck  of  the  flask  a 
double  perforated  rubber  stopper,  provided  with  a  Reitmaier  bulb 
and  a  thistle  tube.  The  end  of  the  latter  should  reach  nearly  to  the 
bottom  of  the  flask.  A  long  strip  of  red  litmus  paper  should  be  sus- 
pended from  the  neck  of  the  flask  and  should  extend  down  into  the 
liquid.     Connect  the  free  end  of  the  Reitmaier  bulb  with  a  condenser.. 


260  PHYSIOLOGICAL  CHEMISTRY. 

The  lower  end  of  the  condenser  is  connected  with  a  bent  tube  which 
extends  down  into  the  liquid  of  the  receiving-  flask.  [Fig.  7).  This 
should  have  about  500  c.c.  capacity  and  contains  50  c.c.  of  ft  oxalic 
acid,  which  will  unite  with  the  NH3  that  will  be  distilled  off.  When 
all  is  in  readiness  pour  into  the  flask,  through  the  tube,  strong  NaOH 
solution  (1:  2)  until  the  liquid  is  decidedly  alkaline.  About  50 — 60  c.c. 
will  be  required.  Heat  the  large  flask  and  distil  over  about  200  c.c. 
Then  replace  the  receiver  by  a  flask  containing  10  c.c.  of  ft  oxalic 
acid  and  some  water  and  continue  the  distillation  till  about  100  c.c. 
of  distillate  passes  over. 

To  each  of  the  receivers  now  add  a  few  drops  of  alcoholic 
rosolic  acid  and  titrate  with  ft  NaOH  to  a  deep  pink  color.  The 
second  flask  serves  as  a  check  and  should  be  free,  or  nearly  free, 
from  ammonia.  The  difference  between  the  number  of  c.c.  of  oxalic 
acid  employed  and  the  number  of  c.c.  of  ft  NaOH  necessary  to  neu- 
tralize the  distillate  gives  the  number  of  c.c.  of  ft  NH3  given  off  in 
the  distillation. 

A  blank  experiment  with  15  c.c.  of  H2S04  and  the  other  reagents, 
but  with  no  urine  added,  should  be  carried  out  exactly  in  the  same 
manner  as  described  above  with  urine,  in  order  to  ascertain  the 
amount  of  ammonia  formed  from  the  nitrogen  that  may  possibly  be 
present  in  the  reagents.  The  number  of  c.c.  of  ft  NH3  thus  found 
should  be  subtracted,  as  a  correction,  from  the  total  number  of  c.c. 
of  ft  NH3  given  off  in  the  experiment  with  urine.  The  difference 
represents  the  number  of  c.c.  of  ft  NH3  formed  from  the  nitrogen 
actually  present  in  the  urine.  This  difference  multiplied  by  the  ft 
factor  of  nitrogen  (0.0014)  gives  the  amount  of  nitrogen  contained  in 
5  c.c.  of  urine.  The  amount  of  total  nitrogen  present  in  100  c.c.  or  in 
the  24  hours'  urine  can  be  readily  calculated. 

2. — *  Dumas'  method. — Five  c.c.  or  more  of  the  urine  are 
acidulated  with  sulphuric  acid  and  evaporated  to  dryness 
in  a  copper  boat.  This  is  then  filled  with  copper  oxide  and 
placed  in  a  combustion  tube  and  heated.  The  nitrogen 
present  is  set  free  and  is  collected  in  a  suitable  apparatus 
and  measured.  From  this,  the  amount  of  nitrogen  in  the 
total  urine  can  be  calculated. 

3. — ^Varrentrapp- Will's  method. — This  gives  good  results 
but  it  is  more  expensive  and  takes  more  time  than  the 
Kjeldahl  method  and  hence  has  been  supplanted  by  it. 
The  residue  from  5  c.c.  of  urine  is  mixed  with  soda-lime 


QUANTITATIVE  ANALYSIS.  261 

and  ignited.  The  nitrogen  present  is  converted  into  am- 
monia and  this  is  estimated  by  receiving  it  into  ft  acid 
solution. 

4. — Liebig's  method  for  titration  of  urea  gives  results 
which  correspond  very  closely  to  the  total  nitrogen  present, 
rather  than  to  urea  itself.     (See  p.  251). 


XIII.     Determination  of  Albumin  (and  Globulin). 


1. — Scherer's  method. — Place  100  c.c.  of  the  clear  urine  in  a  small 
beaker.  If  much  albumin  is  present,  take  less  and  dilute.  If  the 
reaction  is  not  distinctly  acid,  acidulate  slightly  with  acetic  acid  and 
heat  on  the  water-bath  for  about  %  hour.  The  beaker  should  be 
covered  and  so  placed  that  the  lower  half  containing  the  urine  passes 
through  the  ring  of  the  water-bath.  Heat  till  a  coagulum  of  coarse 
floccules  forms.  The  addition  of  a  minute  amount  of  acid  aids  this, 
but  a  larger  amount  must  be  avoided.  (Compare  Exp.  8,  p.  41;  also 
Exp.  3.  p.  110;.  Filter  through  a  weighed  filter,  previously  dried  at 
120 — 130%  and  wash  with  hot  water  till  the  precipitate  ceases  to  give 
the  chloride  reaction.  Fill  the  funnel  several  times  with  absolute 
alcohol,  then  twice  with  ether  (to  remove  fats),  and  dry  at  120—130° 
till  the  weight  is  constant. 

It  is  well  to  make  a  few  preliminary  tests  on  the  completeness 
of  coagulation.  For  this  purpose  test  the  reaction  and  if  acid,  place 
some  of  the  urine  in  a  test-tube  and  heat  in  a  water-bath  till  coagu- 
lated, then  for  a  few  minutes  in  the  flame  and  filter.  If  the  filtrate 
is  cloudy  or  on  testing  with  acetic  acid  and  potassium  f  errocyanide 
gives  a  cloudiness,  the  urine  is  not  sufficiently  acid  and  a  drop  or  so 
of  acetic  acid  must  be  added.  In  this  way  make  one  or  more  tests 
till  complete  precipitation  is  obtained,  and  then  acidulate  to  the 
proper  degree  the  amount  of  urine  taken  for  the  estimation. 

Bearing  in  mind  the  fact  that  NaCl  favors  the  coagulation  of 
albumin  with  acetic  acid  (Exp.  8,  p.  41),  it  is  well  to  add  to  the  urine 
which  has  been  measured  out  10 — 15  per  cent,  of  common  salt. 

2. — *Denfiimetric  metjtod. — In  this  method  the  specific 
gravity  of  the  urine  before  and  after  coagulation  is  deter- 
mined and   the   difference   between   the  specific   gravities 


262  PHYSIOLOGICAL,  CHEMISTRY. 

multiplied  by  400  (Zahor's  factor)  gives  the  number  of  grams 
of  albumin  in  100  c.c. 

The  amount  of  acetic  acid  necessary  to  coagulate  is 
ascertained  as  given  above.  This  amount  is  then  added  to 
the  urine  and  the  specific  gravity  determined.  The  coagu- 
lation should  be  carried  on  in  such  a  manner  that  no  water 
is  lost  by  evaporation,  and  for  that  purpose  the  urine  is 
placed  in  a  bottle  provided  with  a  rubber  stopper  and 
clamp  (beer  bottle).  This  is  now  immersed  in  water  and 
heated  to  boiling  for  10 — 15  minutes.  The  flask  is  then 
cooled,  the  contents  filtered  and  the  specific  gravity  of  the 
filtrate  determined. 

The  urinometers  employed  should  read  to  four  decimal 
places,  or  better  still,  the  specific  gravity  should  be  deter- 
mined by  means  of  a  picnometer.  The  results  are  excellent 
and  approach  closely  the  gravimetric  results. 

3. — *  Roberts- Stolnikoff's  method, — A  solution  containing 
3^  mg\  of  albumin  in  100  c.c.  gives  a  faint  but  distinct 
reaction  with  Heller's  test  in  2 — 3  minutes.  Hence  dilute 
the  urine  under  examination  till  it  gives  the  test  under 
these  conditions.  Then  calculate  the  amount  of  albumin 
present  in  the  24  hours'  urine.  The  results  are  fairly  good 
for  clinical  purposes. 

4. — "Esbach's  albuminometer. 

Reagent, — 10  g.  of  picric  acid  and  20  g.  of  citric  acid 
are  dissolved  in  1  liter  of  water. 

Execution.—  The  instrument  is  filled  with  urine  to  the  U 
mark,  then  with  the  reagent  to  the  mark  R.  The  tube  is 
closed  with  a  stopper,  inverted  several  times  and  then  set 
aside  in  an  upright  position  for  24  hours.  Read  off  the 
height  of  the  precipitate  in  the  tube.  The  figures  indicate 
grams  of  albumin  in  one  liter. 

If  much  albumin  is  present  the  urine  should  first  be 
diluted.     The  specific  gravity  should  be  1.010  or  lower. 


QUANTITATIVE   ANALYSIS.  263 

The  results  obtained  by  this  method  are  only  roughly 
approximate.  The  method  possesses  the  same  value  as 
Einhorn's  saccharimeter. 


XIV.     ^Separate  Determination  of  Globulin  and  Albumin. 

1. — Hammarsten's  method. — Measure  out  25 — 100  c.c.  of 
the  urine,  according-  to  the  amount  of  albumin  present  and 
place  in  a  beaker.  Render  slightly  alkaline  with  potas- 
sium hydrate,  filter  to  remove  phosphates  and  wash.  Add 
the  wash-water  to  the  filtrate.  Neutralize  with  acetic  acid. 
For  100  c.c.  of  solution  add  120  g.  of  pulverized  magnesium 
sulphate,  warm  to  30°  and  stir  well;  then  set  aside  for  24 
hours  in  the  cold.  Filter  through  a  weighed  filter,  pre- 
viously dried  at  110°;  rinse  out  the  beaker  with  saturated 
magnesium  sulphate  solution  and  wash  with  the  same  till 
the  filtrate  ceases  to  give  a  reaction  for  albumin  on  heating 
with  acetic  acid.  Then  dry  the  precipitate  for  several 
hours  at  110°  to  coagulate  the  globulin,  cool  and  wash  with 
hot  water  till  all  magnesium  sulphate  is  removed.  Then 
wash  with  alcohol  and  ether,  dry  at  110°  till  the  weight 
is  constant,  cool  and  weigh.  Subtract  the  weight  of  the 
filter;  the  difference  represents  the  weight  of  the  globulin 
together  with  some  inorganic  matter.  Hence,  ignite  the 
filter  and  weigh  the  ash.  Subtract  the  weight  of  the  ash 
from  the  weight  of  the  globulin  as  first  found;  this  gives 
the  weight  of  the  globulin.  Calculate  the  amount  in  the 
24  hours'  urine. 

If  the  globulin  and  albumin  are  determined  by  Scherer's 
method,  the  difference  between  the  two  results  gives 
albumin. 

2. — PohVs  method. — In  this  the  globulin  is  precipitated 
with  an  equal  volume  of  a  saturated  solution  of  ammonium 
sulphate    and    the   precipitate    is    washed    with   a   semi- 

18 


264  PHYSIOLOGICAL    CHEMISTRY. 

saturated  solution  of  the  same.     After  that  the  process  is 
the  same  as  that  given  above. 

The  results  are  about  the  same  as  those  obtained  by 
Hammarsten's  method;  moreover,  the  method  possesses  the 
advantage  that  the  filter  does  not  clog. 


XV.     ^Determination  of    Albumose. 

From  50  to  100  c.c.  of  the  urine,  according  to  the  amount 
of  albumose  present,  are  taken  for  this  purpose.  Albumin 
and  globulin,  if  present,  must  first  be  removed  by  the  aid 
of  acetic  acid  and  heat.  To  facilitate  the  precipitation 
10  g.  of  NaCl  can  be  added;  the  liquid  in  that  case  must  be 
filtered  boiling  hot,  and  the  precipitate  washed  with  boil- 
ing water.  The  filtrate  and  wash-water  are  concentrated 
to  about  the  original  volume,  cooled  and  strongly  acidu- 
lated with  sulphuric  acid,  then  treated  with  an  acid  solu- 
tion of  sodium  phosphotungstate  (1  volume  of  dilute 
H2S04  and  3  volumes  of  sodium  phosphotungstate  solution) 
as  long  as  a  precipitate  forms.  The  precipitate  is  filtered 
and  washed  with  dilute  sulphuric  acid  (1:3);  the  filter  with 
its  contents  is  placed  in  a  flask  and  the  nitrogen  determined 
by  the  Kjeldahl  method.  The  proper  allowance  for  the 
blank  is  made.  The  weight  of  nitrogen  multiplied  by  6.25 
gives  the  weight  of  the  albumose. 


QUANTITATIVE    ANALYSIS.  265 


Average  Composition  of  Urine  (after  SCHOTTEN). 

Average    quantity  per  24   hours   is   placed   at   about 
1500  c.c. 

I.  Normal  Constituents. — 60  g. 

A. — Organic, — 35  g. 

Urea,  30  g. 

Uric  acid,  0.6  g. 

Creatinin,  0.8  g. 

Xanthin  compounds,  oxalic  acid,  oxaluric  acid, 
volatile  fatty  acids,  lactic  acid,  glycerin- 
phosphoric  acid,  sulphocyanic  acid. 

Hippuric  acid;  phenol-,  cresol-,  pyrocatechin-, 
indoxyl-  and  skatoxyl-sulphuric  acids;  oxy- 
phenylacetic  acid;  hydrocumaric  acid. 

Urine  pigments. 

Ferments;  sulphur  and  nitrogen  containing  sub- 
stances; substances  of  unknown  composition 
free   from   sulphur  and   nitrogen.      Total   a 
few  grams. 
B — Inorganic, — 25  g. 

Sodium  chloride,  15  g. 

Sulphuric  acid,  2.5  g. 

Phosphoric  anhydride,  2.5  g. 

Nitric  acid,  0.1  g.  (or  less). 

Potassium  oxide,  3.0  g. 

Ammonia,  0.7  g. 

Magnesium  oxide,  0.5  g. 

Calcium  oxide,  0.3  g. 

Iron,  0.01  g.  (or  less). 

II.  Abnormal,  or  Pathological  Constituents. 

Albumin,  ,5-Oxybutyric  acid, 

Globulin,  Blood,  blood  pigments, 

Albumose,  Melanin  and  other  pigments, 

Pepton.  ?  Bile,  pigments  and  acids, 

Mucin,  Fat,  cholesterin,  lecithin, 

Grape  sugar,  Leucin,  tyrosin, 

Milk  sugar,  Alkapton     (uroleucinic     and 

Laevulose,  homogentisinic  acids). 

Inosite,  Cystin,  putrescin,  cadaverin, 

Aceton,  acetacetic  acid,  Hydrogen  sulphide. 


266 


PHYSIOLOGICAL  CHEMISTRY. 


Analysis  of  24  Hours'  Urine. 


Volume,  c.c 

Specific  gravity 

Total  solids 

Water 

Organic  substances 

Inorganic  substances 

Total  nitrogen 

Urea 

Uric  acid 

Creatinin 

Ammonia 

Hippuric  acid 

Phenol 

Potassium  oxide 

Sodium  oxide 

Sodium  chloride 

Calcium  oxide 

Magnesium  oxide 

Chlorine 

Phosphoric  anhydride  (P205) 

Sulphuric  anhydride  (S03) 

Sulphuric  acid  from  sulphur  containing  or 
ganic  substances 


Man. 


Meat. 

Bread. 

1672 

1920 

2055 
1  046 

248.244 

1806 . 755 

198 . 061 

50.183 

65  34 

67.2 

20.6 
0.253 
0.961 

1.398 
2.163 

traces 

0  357 

15.597 

2.445 

3.308" 

1.314 
3.923 

3.991 

27  126 

0.328 
0.294 

0.339 
0.139 
4.996 
1.658 
1.265 

5.713 

3.817 

3.437 

4.674 

0.2199 
10.299 

3.165 

Horse. 


The  results  given  in  the  first  and  second  columns  were  obtained 
by  Bunge  from  the  urine  of  the  same  individual  under  exclusive  meat 
and  vegetable  diet;  each  column  represents  the  analysis  of  a  single 
24  hours'  urine.  The  analytical  results  in  the  third  column  are  those 
of  E.  Salkowski. 


QUANTITATIVE    ANALYSIS. 


267 


In  the  following-  table  are  given  the  averages  of  the  results 
obtained  from  the  analysis  of  normal  24  hours'  urine  of  students  at 
the  University  of  Michigan: 


Volume 

Specific  gravity 

Urea 

Uric  acid - 

Sodium  chloride 

Sulphuric  acid  (S03). . . 
Phosphoric  acid  (P205 


Men 

Women. 

1120 

(333)* 

1000             (56) 

1.022 

(334) 

1.020      (56) 

30.10 

(320) 

21.54        (48) 

.55 

(266) 

.42        (36) 

13.93 

(275) 

11.19        (42) 

2.30 

(266) 

2.11        (40) 

2.16 

:jiis, 

1.78        (44) 

*The  figures  in  brackets  refer  to  the  number  of  determinations 


made. 


Milk  Analysis. 


The  milk  to  be  analyzed  should  be  thoroughly  shaken  just  before 
each  portion  is  taken  for  analysis,  in  order  to  insure  a  true  sample. 
The  quantity  to  be  taken  is  measured  out  by  means  of  a  clean  and 
dry  10  c.c.  pipette  graduated  in  TV  c.c.  The  quantity  of  milk  taken 
multiplied  by  the  specific  gravity  gives  the  weight  of  the  milk 
employed  for  the  determination. 

1. — Specific  gravity. — Determine  the  specific  gravity  of  the  sam- 
ple, at  15%  by  means  of  : 

(a).  The  pienometer,  or  specific  gravity  bottle.  This  is  done  in 
the  following  manner:  The  pienometer  is  cleaned,  dried  and  weighed. 
It  is  then  filled  with  distilled  water  at  15'  and  weighed  again:  the 
difference  is  the  weight  of  'a  certain  volume  of  water.  The  instru- 
ment is  then  dried,  filled  with  the  sample  at  15"  and  weighed  again 
and  the  weight  of  the  same  volume  of  milk  is  thus  obtained.  The 
weight  of  the  milk  divided  by  the  weight  of  the  same  volume  of 
water  gives  the  specific  gravity. 

lb).  The  lactometer. — There  are  two  forms  of  this  instrument  in 
common  use.  The  Quevenne-Muller  lactometer,  employed  largely  in 
Europe,  gives  the  specific  gravity  direct.  The  lactometer  of  the 
New  York  Board  of  Health  reads  fromO',  the  density  of  water  at 
15    and  which  corresponds  to  1.0,  to  120    which  represents  a  specific 


268  PHYSIOLOGICAL  CHEMISTRY. 

gravity  of  1.0348.  100°  on  this  scale  represents  the  specific  gravity 
of  1.029,  which  is  taken  as  the  minimum  density  of  genuine  milk.  In 
the  absence  of  a  lactometer  the  ordinary  urinometer  may  be  used, 
although  the  divisions  are  very  small  and  the  reading,  consequently, 
is  not  accurate. 

Place  a  sample  of   the  milk  in  a  suitable   cylinder  or  50  c.c 
graduate   and  determine   the   density.     Compare  the  reading  thus 
obtained  with  the  specific  gravity  as  given  by  an  ordinary  lactometer. 

Determine  the  specific  gravity  of  the  skimmed  milk  obtained 
from  the  following  experiment.     What  is  the  result? 

What  is  the  effect  of  the  addition  of  water  to  milk?  To 
skimmed  milk?    What  is  the  effect  of  the  removal  of  cream? 

2.— Creamometer—  Fill  a  50  c.c.  graduate  to  the  mark  with  milk, 
cork  and  set  aside  for  24  hours  at  the  ordinary  room  temperature. 
Note  the  volume  of  the  cream  and  calculate  the  volume  per  cent.  A 
good  milk  should  give  10 — 12  per  cent,  of  cream. 

Remove  the  skimmed  milk  from  below  the  layer  of  cream  by 
means  of  a  pipette  and  determine  the  density  according  to  16. 

3. — Total  solids. — Place  2  c.c.  of  milk  in  a  previously  weighed 
watch-glass  and  evaporate  on  the  water-bath  to  dryness.  Then  wipe 
the  bottom  of  the  watch-glass  and  place  it  in  an  air-bath  at  100—105° 
for  3  hours.  Cool  in  the  desiccator  and  weigh  rapidly.  Calculate  the 
per  cent,  of  total  solids.  This  result  subtracted  from  100  gives  the 
per  cent,  of  water. 

i. — Ash. — Place  5  c.c.  of  milk  in  a  previously  weighed  porcelain 
crucible  and  evaporate  to  dryness  on  the  water-bath.  Then  carefully 
ignite  so  as  to  char  the  mass  slowly  and  thus  avoid  spurting.  The 
ignition  must  be  continued  till  the  ash  is  grayish-white  and  free  from 
carbon.  Cool  in  the  desiccator  and  weigh.  Calculate  the  per  cent. 
of  ash. 

5. — Fat. — Roll  up  into  a  coil  a  strip  of  thick  filter  paper  about  2 
inches  wide  and  24  inches  long,  and  tie  it  with  a  thread  or  wire.  Five 
c.c.  of  milk  are  allowed  to  run  slowly  onto  one  end  of  the  coil.  The 
coil,  dry  end  down,  is  placed  on  a  watch-glass  and  dried  in  an  air-bath 
at  100 — 105°  for  one  hour,  or  longer  if  necessary.  It  is  then  placed  in 
a  Soxhlet  extraction  apparatus  which  is  connected,  by  means  of 
sound,  well  fitting  corks,  to  an  inverted  condenser  above,  and  to  a  150 
c.c.  wide  neck,  round,  or  Erlenmeyer  flask  below  (Fig.  8).  The  weight 
of  the  clean,  dry  flask  is  first  ascertained.  By  means  of  a  small  fun 
nel  pour  ether  into  the  apparatus  from  above,  until  it  siphons;  then 


QUANTITATIVE    ANALYSIS. 


269 


add  about  half  as  much  more  ether.  The  flask  is  now  heated,  cau- 
tiously, on  a  water-bath  so  that  the  ether  will  siphon  about  every  five 
minutes.  The  extraction  will  be  completed  in  from  1  to  1)4  hours. 
Then  remove  the  paper  coil  and  continue  heating  till  the  ether  fills 
the  extraction  apparatus,  and  is  almost  ready  to  siphon.  Disconnect 
the  flask  and  trans- 
fer the  ether  from 
the  apparatus  to  a 
bottle.  The  flask 
still  contains  some 
ether.  Heat  on  the 
water-bath  to  dry- 
ness, wipe  care- 
fully, and  finally 
dry  in  an  air-bath 
at  100—105°  for  one 
hour.  Cool  in  a 
desiccator  or  in 
the  balance  case 
and  weigh.  Calcu- 
late the  per  cent, 
of  fat.  Subtract 
this  result  from 
that  of  total  solids; 
the  difference  is 
solids  not  fat. 

In  working 
with  ether  great 
care  must  be  taken 
to  prevent  acci- 
dents. The  corks 
must  be  large 
enough  to  fit  snug- 
ly, and  the  gas  must  be  turned  oft'  until  everything  is  in  readiness 
to  begin  the  extraction.  To  obtain  very  accurate  results  the  coil  of 
paper  should  be  first  extracted  with  ether  to  remove  what  fat  may  be 
present.     Paper,  from  which  fat  has  been  removed,  can  be  purchased. 

6. — Lactose. — Place  about  380  c.c.  of  water  in  a  beaker,  add  20  c.c. 
of  milk  and  mix  thoroughly.  Then  add  gradually  about  2  c.c.  of  dilute 
acetic  acid  (1 :  10),  with  constant  stirring,  till  a  flocculent  precipitate 
forms.  The  reaction  should  be  distinctly  acid.  Place  the  beaker  on 
a  wire  gauze  and  heat  to  boiling  for  }i  hour;  then  filter  through  a 


Fig.  8. 


270  PHYSIOLOGICAL  CHEMISTRY. 

wet  filter.  Rinse  the  beaker  several  times  with  hot  water,  and  finally 
wash  the  residue  on'the  filter,  proteids  and  fat,  with  hot  water.  Con- 
centrate the  combined  filtrate  and  wash-water  to  about  150  c.c.  Cool 
and  dilute  in  a  200  c.c.  measuring"  flask  to  the  mark.  Determine  the 
lactose  in  this  solution  with  Fehling's  solution  according  to  the 
method  described  on  p.  247. 

10  c.c.  of  Fehling's  solution  corresponds  to  0.067  g.  of  milk  sugar. 

Calculate  the  per  cent,  of  lactose. 

7. — Casein. — To  50  c.c.  of  water  in  a  small  beaker  add  10  c.c.  of 
milk  and  mix.  Warm  on  the  water-bath  to  40°,  then  add  2%  c.c.  of 
potassium  alum  solution  (1:10)  and  stir  thoroughly.  A  finely  floccu- 
lent  precipitate  forms  which  should  settle  rapidly  and  the  liquid 
should  be  clear.  Let  stand  for  about  15  minutes  at  40°,  then  filter. 
If  the  filtrate  is  cloudy  pass  it  again  through  the  filter.  Wash 
several  times  with  water.  Reserve  the  combined  filtrate  and  wash- 
water  for  the  next  determination. 

Place  the  filter  with  its  contents  in  a  Kjeldahl  flask  of  about  250 
c.c.  capacity,  and  treat  exactly  according  to  the  method  given  for 
total  nitrogen  in  urine,  p.  258. 

A  blank  experiment  with  a  filter  paper  and  all  the  reagents 
should  be  carried  through  in  exactly  the  same  manner  in  order  to 
ascertain  how  much  NH3  may  be  given  off  by  the  reagents  them- 
selves. The  number  of  c.c.  of  xrr  NH3  thus  found  should  be  subtracted 
as  a  correction  from  the  total  number  of  c.c.  of  T%-  NH3  given  off  in 
the  determination.  The  difference  multiplied  by  the  j-q  factor  of 
nitrogen,  0.0014,  gives  the  amount  of  nitrogen  contained  in  the  casein 
precipitated  from  10  c.c.  of  milk.  This  amount  of  nitrogen  multi- 
plied by  6.37  gives  the  amount  of  casein  in  10  c.c.  of  milk.  Calculate 
the  per  cent,  of  casein. 

8. — Globulin  and  albumin. — The  filtrate  from  the  alum  precipitate 
of  casein  in  the  preceding  experiment  contains  globulin  and  albu- 
min. To  this  filtrate  add  10  c.c.  of  Almen's  tannic  acid  solution. 
Filter  off  the  voluminous  precipitate  of  proteids,  wash  several  times 
with  water,  and  allow  to  drain.  Place  the  filter  and  contents  in  a 
Kjeldahl  flask  and  determine  the  nitrogen  as  above,  making  the 
proper  correction  for  a  blank.  The  amount  of  nitrogen  found  multi- 
plied by  the  factor  6.37  gives  the  amount  of  albumin  and  globulin  in 
10  c.c.  of  milk.     Calculate  the  per  cent,  of  albumin  and  globulin. 

Almen's  tannic  acid  solution  is  prepared  according  to  the  follow- 
ing formula:  4  g.  of  tannic  acid;  8  c.c.  of  25  per  cent,  acetic  acid; 
90  c.c.  of  90  per  cent,  alcohol;  100  c.c.  of  water. 


QUANTITATIVE    ANALYSIS. 


271 


9.— Total  nitrogen  in  milk. — Place  10  c.c.  of  milk  in  a  Kjeldahl 
flask,  add  15  c.c.  of  H2S04  and  treat  as  above  under  Exp.  7.  This  gives 
the  total  nitrogen  present  in  the  milk  and  serves  as  a  control  on  the 
two  preceding  determinations. 

A  report  of  the  results  obtained  is  to  be  made  out,  together  with 
a  statement  as  to  whether  the  milk  is  adulterated  or  not: 


1. 
2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 
10. 
11. 
12. 


Specific  gravity,  whole  milk. . . . 
Specific  gravity,  skimmed  milk. 

Cream,  volume,  per  cent 

Water 

Total  solids 

Solids  not  fat 

Fat 


Ash 

Lactose 

Casein 

Globulin  and  albumin 

Total  nitrogen  as  proteid. 


To  decide  upon  the  purity  of  a  milk  the  determinations  given 
under  1,  5,  and  7  (4  and  6  by  difference),  are  as  a  rule  sufficient.  In 
case  of  doubt  the  ash  may  be  determined.  The  legal  standard  of 
milk  varies  in  different  states.  In  New  York  the  minimum  of  total 
solids  allowed  is  12  per  cent.;  of  fat  3  per  cent.  In  Massachusetts  the 
total  solids  must  not  fall  below  13  per  cent.  New  Jersey  allows  a 
minimum  of  12  per  cent,  for  total  solids. 

The  following  table,  compiled  from  Konig,  shows  the  average 
percentage  composition  of  various  milks: 


Milk  of 


Woman 

Cow 

Cow  (colostrum) 

Goat 

Sheep  

Mare 

Ass 

Hog 

Dog 


"i  i 

3    . 

>> 

a 

oJ 

■Hit1? 

.    CO    tfj 

ft  Go 

u 

-t-> 

"v 

03 

0 
0 

fe 

GO 

Q 

< 

pE| 

J 

107 

1.027 

87.41 

1.03 

1.26 

3.78 

6.21 

793 

1.0315 

87.17 

3.02 

0.53 

3.69 

4.88 

42 

74.67 

4.04 

13.60 

3.59 

2.67 

38 

85.71 

3.20 

1.09 

4.78 

4.46 

32 

1.034 

80.82 

4.79 

1.55 

6.86 

4.91 

47 

1.0347 

90.78 

1.24 

0.75 

1.21 

5.67 

4 

89.64 

0.67 

1.55 

1.64 

5.99 

7 

84.04 

7. 

23 

4.55 

3.13 

28 

75.44 

6.10 

5.05 

9.57 

3.00 

< 


0.31 
0.71 
1.56 
0.76 
0.89 
0.35 
0.51 
1.05 
0.73 


272  PHYSIOLOGICAL  CHEMISTRY. 


Quantitative  Analysis  of  Gastric  Juice. 

Toepfer's  method. — The  following'  reagents  are  necessary: 

1. — Deci-normal  sodium  hydrate  solution. 

2. — A  1  per  cent,  alcoholic  solution  of  phenol-phthalei'n.  This 
indicates  total  acidity. 

3. — A  1  per  cent,  aqueous  solution  of  sodium  alizarin-sulphonic 
acid.  This  indicates  all  acids  except  the  loosely  combined  hydro- 
chloric. 

•4. — A  0.5  per  cent,  alcoholic  solution  of  di-methyl  amido-azoben- 
zol.     This  indicates  only  free  HC1. 

The  method  is  as  follows:  Measure  out  into  each  of  three  beak- 
ers 10  c.c.  of  the  filtered  gastric  juice.  If  necessary  a  smaller  amount 
may  be  taken,  or  the  gastric  juice  may  be  diluted. 

To  beaker  No.  1  add  1 — 2  drops  of  phenol-phthalei'n,  then  run  in 
the  deci-normal  sodium  hydrate,  not  to  the  first  pink  color,  but  to  a 
dark  red  which  no  longer  increases  in  intensity.  Note  the  number  of 
c.c.  of  reagent  employed. 

To  beaker  No.  2  add  3 — 4  drops  of  the  alizarin  solution  and  then 
titrate  with  deci-normal  sodium  hydrate  till  the  first  pure  violet  color 
is  reached.     Note  the  number  of  c.c.  employed. 

To  beaker  No.  3  add  3 — 4  drops  of  di-methyl  amido-azobenzol 
solution.  A  yellow  color  indicates  the  absence  of  free  HC1.  If  a  red 
color  is  present  run  in  deci-normal  sodium  hydrate  till  it  just  disap- 
pears.    Note  the  number  of  c.c.  employed. 

The  number  of  c.c.  required  for  beaker  No.  1  gives  the  total 
acidity. 

The  difference  between  the  number  of  c.c.  of  deci-normal  sodium 
hydrate  required  for  beaker  No.  1  (total  acids)  and  the  number 
required  for  beaker  No.  2  (all  acids  except  loosely  combined),  gives 
the  loosely  combined  HC1. 

The  number  of  c.c.  required  for  beaker  No.  3  gives  the  free  HCL 

The  number  of  c.c.  of  reagent  required  for  beaker  No.  2  minus 
the  number  of  c.c.  required  for  beaker  No.  3,  gives  the  organic  acid 
and  acid  salts. 

The  student  will  receive  two  unknowns,  containing  free  and 
loosely  combined  hydrochloric  acid  and  an  organic  acid.    In  reporting 


QUANTITATIVE    ANALYSIS.  273 

the  results,  give  the  number  of  c.c.  of  &  alkali  that  would  be  required 
to  neutralize  each  of  the  acids  in  100  c.c. 


Blood. 

The  examination  of  blood  is  of  very  great  clinical  im- 
portance and  every  student  should  make  himself  familiar 
with  the  methods  that  are  usually  employed.  The  examin- 
ation from  the  physical  and  chemical  standpoint  embraces 
a  determination  of  the  specific  gravity;  counting  of  red  and 
of  white  blood  cells;  and  a  determination  of  the  amount  of 
haemoglobin  present. 

1. — * Specific  gravity. — In  the  case  of  blood  from  a  patient 
it  is  not  possible  to  determine  the  density  according  to  the 
methods  described  under  urine  and  milk.  A  much  smaller 
amount  must  suffice.  If  a  drop  of  blood  is  introduced  into 
a  mixture  of  chloroform  and  benzol  it  will  not  mix  but  will 
either  float  on  the  surface,  or  sink  to  the  bottom,  or  remain 
in  the  body  of  the  liquid  where  it  is  placed.  If  the  drop 
floats  it  is  because  the  liquid  is  heavier  and  in  that  case 
benzol  is  to  be  added,  a  few  drops  at  a  time  and  mixed  by 
means  of  a  rod,  till  the  drop  remains  wherever  it  is  placed. 
If  on  the  other  hand  the  drop  falls  to  the  bottom  it  shows 
that  the  liquid  is  too  light  and  chloroform  is  then  to  be 
added  in  portions  as  in  the  case  of  benzol,  till  the  drop 
remains  stationary  wherever  placed.  In  the  latter  case  the 
drop  of  blood  has  the  same  specific  gravity  as  the  liquid  in 
which  it  floats.  The  specific  gravity  of  the  mixture  is 
therefore  taken  and  this  represents  the  density  of  the  blood. 
The  ordinary  urinometer  may  often  be  used,  if  graduated  to 
about  1.050,  when  the  specific  gravity  is  low.  It  is  better 
to  have  one  that  will  read  from  1.040  to  1.070.  The  meas- 
uring cylinder  which  receives  the  mixture  should  be  abso- 
lutely clean  and  dry.  Several  separate  drops  of  blood  may 
be  advantageously  employed.     The  mixture  of  chloroform 


"274  PHYSIOLOGICAL  CHEMISTRY. 

and  benzol  should  at  the  beginning-  have  a  density  of  about 
1.059,  which  is  that  of  normal  blood. 

Inasmuch  as  the  specific  gravity  of  blood  is  subject  to 
variation,  not  so  much  because  of  differences  in  the  density 
of  the  plasma,  but  because  of  differences  in  the  number  of 
cells  and  hence  the  amount  of  haemoglobin  present,  it  fol- 
lows that  this  method  can  also  be  used  to  determine  ap- 
proximately the  amount  of  haemoglobin  present. 

2. — * Determination  of  hcemoglobin. — This  can  be  deter- 
mined very  accurately  by  means  of  Hiifner's  spectrophoto- 
meter, but  this  instrument  is  not  used  for  clinical  purposes. 
Among  the  clinical  methods  may  be  mentioned  the  follow- 
ing: 

(a).  From  specific  gravity  oj  blood. — From  tables  espe- 
cially prepared  the  amount  of  haemoglobin  present  can  be 
readily  ascertained.  A  specific  gravity  of  1.059  (normal 
blood)  represents  100  per  cent,  of  haemoglobin.  Ham- 
merschlag's  tables,  as  given  in  Cabot's  "Examination  of 
Blood,"  can  be  used  for  this  purpose.  The  method  is  not 
applicable  in  cases  of  dropsy. 

(b).  FleischVs  hcemometer. — In  this  method  a  minute, 
definite  quantity  of  blood  is  transferred  to  water  and  the 
color  of  this  solution  is  compared  with  that  of  a  wedge  of 
colored  glass.  When  the  colors  match  the  per  cent,  of 
haemoglobin  is  read  off  on  the  scale;  100  corresponds  to  the 
color  of  the  normal  blood  of  man,  about  90  corresponds  to 
that  of  woman.  This  instrument,  although  it  gives  only 
approximate  results,  is  the  one  most  commonly  employed. 

(c).  Hoppe-Seyler's  colorimetric  method. — In  this  method 
the  blood  is  treated  with  carbon  monoxide  and  the  color  of 
the  resulting  carbon  monoxide  haemoglobin  solution  is  com- 
pared with  a  solution  of  pure  carbon  monoxide  haemoglobin 
of  known  strength.  The  two  colors  can  therefore  be 
matched  perfectly,  which  is  not  always  the  case  in  the 


QUANTITATIVE    ANALYSIS. 


275. 


preceding  method.  Still  greater  accuracy  is  attained  by 
eliminating-  the  partition  that  exists  between  the  two  com- 
partments by  means  of  an  Albrecht  glass  cube.  In  that 
case  the  colors  are  compared  side  by  side  as  in  a  Soleil- 
Ventzke  saccharimeter.     Although  the  apparatus  is  some- 


Fig.  9. 

what  expensive  it  can  be  easily  manipulated  and  gives 
much  more  accurate  results  than  either  of  the  preceding 
(Fig.  9). 

Recrystallized  haemoglobin  from  the  dog,  or  horse  is 
employed.  A  strong  solution  (2 — 3  per  cent.)  of  this  haemo- 
globin is  made  and  treated  with  carbon  monoxide.  In  a 
portion  of  this  solution  the  amount  of  haemoglobin  present 
is  determined  by  evaporating,  drying  and  weighing.  The 
remainder  of  the  solution,  saturated  with  carbon  monoxide, 
is  drawn  up,  in  portions  of   about  6  c. a,  into  glass  tubes. 


276  PHYSIOLOGICAL  CHEMISTRY. 

and  these  are  then  sealed  at  both  ends.  These  solutions 
once  prepared  will  keep  perfectly.  Before  use  the  contents 
of  one  of  the  tubes  is  diluted  with  water  treated  with 
carbon  monoxide  till  the  solution  contains  0.2  per  cent,  of 
CO-haemoglobin. 

The  blood,  obtained  by  puncture,  is  drawn  up  into  a 
graduated  capillary  pipette  such  as  is  used  in  the  Thoma- 
Zeiss  blood  counter.  0.04  to  0.06  c.c.  or  more  of  the  blood  is 
measured  out  and  transferred  to  a  small  graduated  cylinder 
(10  c.c.  graduated  in  0.1  c.c,  stoppered).  The  pipette  is 
carefully  rinsed  several  times  with  distilled  water  and  this 
is  added  to  the  cylinder.  A  drop  of  very  dilute  NaOH  solu- 
tion is  added  and  then  a  current  of  illuminating  gas  is 
passed  through  the  liquid  to  convert  the  pigment  into  CO- 
haemoglobin. 

The  standard  0.2  per  cent.  CO-haemoglobin  solution  is 
placed  in  a  similar  cylinder  and  the  colors  are  compared. 
The  blood  tube  will  have  a  deeper  color,  hence  water  satu- 
rated with  carbon  monoxide  is  added  till  the  two  solutions 
have  approximately  the  same  color.  They  are  then  drawn 
up  into  the  "double  pipette"  and  compared.  Further  ad- 
dition of  water  is  made  to  the  blood  till  on  comparison  it 
has  the  same  color  as  the  standard  0.2  per  cent.  CO-haemo- 
globin  solution.  Now,  note  the  volume  to  which  the  blood 
has  been  diluted  to  obtain  this  color.  Knowing  the  amount 
of  blood  taken  the  per  cent,  of  haemoglobin  can  be  readily 
calculated  from  the  formula: 

T3  ,      ,,  ,  ,  .  0.002  X  v.  X  100 

Per  cent,  of  haemoglobin  —  — 

w 

w  represents  the  weight  (or  volume)  of  the  blood  taken; 
v  represents  the  volume  to  which  this  was  diluted;  0.002 
represents  the  contents  of  the  standard  CO-haemoglobin 
solution.  By  this  method  the  blood  of  normal  individ- 
uals contains  on  an  average  14.08  per  cent,  of  haemoglobin. 
The  results  are  higher  than  those  obtained  by  Fleischl's 


QUANTITATIVE   ANALYSIS.  277 

hcemometer  or  Gower's  hasmoglobinometer.  The  above 
description  of  Hoppe-Seyler's  "double-pipette"  is  given  at 
length  because  this  valuable  instrument  has  not  received 
the  attention  that  it  undoubtedly  merits. 

3. — * Counting  of  corpuscles. — The  Thoma-Zeiss  blood- 
counter  is  commonly  employed  for  this  purpose.  An  excel- 
lent description  of  the  use  of  this  instrument  will  be  found 
in  Cabot's  work  on  "Blood". 

Each  student  will  receive  25  quantitative  unknowns, 
containing  the  substances  indicated  below.  These  are  to 
be  determined,  and  a  report  made  on  blanks  provided  for 
that  purpose. 

Nos.  1  and  2  contain  oxalic  acid. 

Nos.  3  and  4  contain  NaOH. 

Nos.  5  and  6  contain  NaCl  (use  Volhard's  method). 

Nos.  7  and  8  contain  NaCl  (use  Mohr's  method). 

Nos.  9  and  10  contain  S03  (volumetrically). 

Nos.  11  and  12  contain  P,05  (volumetricalty). 

Nos.  13,  14  and  15  contain  glucose. 

No.  16  contains  NaCl,  or  S03,  or  P205  (gravimetrically). 

Nos.  17  and  18  contain  urea  (use  Liebig's  method). 

Nos.  19  and  20  contain  urea  (use  ureometer). 

No.  21  contains  uric  acid  (use  Heintz'  method). 

No.  22   contains    uric    acid   (use    Polin's   or   Ludwig's 

method). 
No.  23  contains  albumin  (gravimetric  method). 
Nos.  24  and  25  contain  free  and  loosely  combined  HC1, 

and  organic  acids  in  gastric  juice  (Toepfer's  method). 

Each  student  will  also  make  a  complete  quantitative 
analysis  of  24  hours'  urine,  and  make  a  report  on  the  same. 


278 


PHYSIOLOGICAL    CHEMISTRY. 


Laboratory  of  Physiological  Chemistry. 


REPORT  ON  QUANTITATIVE  UNKNOWNS. 


Number  of 
bottle. 

Substance  estimated. 

Grams  per 
100  c.c. 

Method  em- 
ployed. 

Dated 189. 


QUANTITATIVE   ANALYSIS. 


279 


Laboratory  of  Physiological  Chemistry. 


REPORT   ON   EXAMINATION  OF   URINE. 


Sample  from Submitted  by . 

Received 189. .;   Reported 


.189. 


PHYSICAL    AND    CHEMICAL     EXAMINATION. 

Total  quantity  for  24  hours 

Specific  gravity Total  solids . . . 

Reaction ;  Equivalent  to. 

Color Odor 

General  appearance 

Sediment,  color ;  quantity 


Albumin 

Glucose 

Urea 

Uric  acid 

Oxalates 

Carbonates 

Chlorides 

Phosphates,  acid. 

"  neutral. . 

"  triple 

Sulphates,  total 

"         conjugate. 


r^H      03 

a 


rt 


Bile,  acids 

"     pigments 

Diazo  reaction.... 

Indoxyl 

Skatoxyl 

Phenol 

Aceton 

Acetacetic  acid. . . 

Ammoniacal  fer- 
mentation  

Hydrogen  sulphide 
fermentation 

Fats,  fatty  acids. . 


d 

Z-t    03 


MICROSCOPIC    EXAJIINATION. 

Unorga  n  ized  sedime  n  t . 

Crystalline Amorphous 

Organized  sediment. 

Epithelial  cells  from Non-pathogenic  bacteria. 

Casts Pathogenic  bacteria 

Blood Yeasts 

Pus Moulds 

Spermatozoa Animal  parasites 

(Signed) 


280  PHYSIOLOGICAL  CHEMISTRY. 


Final  Unknowns. 

Each  student  will  receive  25  urine  "  unknowns."  These 
are  not  pathological  urines,  but  they  do  contain  one  or 
more  of  the  common  abnormal  constituents  of  urine  and 
are  given  to  the  student  as  a  matter  of  exercise  to  review 
and  apply  the  characteristic  tests  employed  in  their 
recognition. 

The  first  15  unknowns  may  contain  one  or  more  of  the  following' 
constituents: 

Sugar,  Uric  acid, 

Albumin,  Urates, 

Bile,  Calcium  oxalate, 

Blood,  Amorphous  phosphates, 

Haemoglobin,  Triple  phosphate, 

Fats,  Calcium  acid  phosphate, 

Pus,  Calcium  carbonate. 
Casts. 

The  next  five  may  contain  in  addition  to  the  above  constituents: 

Hippuric  acid,  Diazo  reaction, 

Albumose,  Indican, 

Pyrocatechin,  Hydrogen  sulphide. 

The  last  five  may  contain  in  addition  to  one  or  more  of  the  pre- 
ceding: 

Urea,  excess  or  deficiency,      Leucin, 
Aceton,  Tyrosin, 

Acetacetic  acid,  Cholesterin. 

The  results  obtained  on  examination  of  these  unknowns  are 
reported,  in  sets  of  five,  on  blanks  provided  for  that  purpose. 


QUANTITATIVE   ANALYSIS.  281 

Laboratory  of  Physiological  Chemistry. 


REPORT  ON  UNKNOWNS. 

No Reaction Sp.  Gr . 

Soluble  constituents 

Sediment 


Xo Reaction Sp.  Gr . 

Soluble  constituents 

Sediment 


No Reaction Sp.  Gr. 

Soluble  constituents 

Sediment 


Xo Reaction Sp.  Gr . 

Soluble  constituents '. 

Sediment 


No Reaction Sp.  Gr. 

Soluble  constituents 

Sediment 


Dated Name . 


CHAPTER     XII. 

TABLES   FOR  EXAMINATION   OF    UPINE.- 

Collect  the  urine  passed  during-  24  hours,  mix  and  measure 
certain  the  and  reaction:  note  the  color,  odor,  and 

general  appearance,  whether  cloudy  or  clear.  Set  a  portion  aside  in 
a  clean  glass  better,  a  conical  one)  and  allow  the  deposit  to 

subside  for  some  hours  for  microscopic  examination,  as  given  in  the 
following  tab".-  Considerable  time  can  be  saved  by 

the  use  of  a  centrifuge.  Filter  another  portion  and  test  the  clear 
filtrate  according  to  table   I 

microscopic  mrAMTWATrpw    yg  -;?.::." ..?.Y  DEPOSIT-. 

er  the  de:  formed,  or  has  been  thrown  out  by  centri- 

fugation  :  _::.    ;.   ircr.   from  '--.   '.:'.- 

torn  of  the  vessel,  place  this  on  a  glass  slide  : '-:  with  a  cover- 
slip  and  examine  under  a  microscope  which  magnifies  from  300  to 
500  diameters.  It  is  best  to  examine  first  with  a  low  power  {%  inch 
objective)  then  with  a  higher  power  {Ve  inch).  For  pathogenic  organ- 
isms a  TV  inch  homogeneous  oil-immersion  objective  is  necessary. 
The  objects  seen  under  the  microscope  may  be  either  crystalline 
amorphous,  or  organized:  any  one.  or  all  three  of  these  groups  may 
be  represented.  The  same  substance  m:-.y  i::-ir  :-_:  :~t  -.-~-  in 
crystals,  and  at  another  in  the  amorphous  form,  and  may  thus  indi- 
cate different  pathological  conditions.  In  applying  reagents  to  the 
sediment  on  a  slide  avoid  leaving  any  of  the  liquid  on  top  of  the 
cover  glass.  A  glass  tube  drawn  out  in  the  middle  and  cut  in  two  will 
furnish  useful  pipettes.  In  order  to  cause  the  reagent  to  go  under 
the  cover-glass  a  piece  of  blotting  or  filter  paper  may  be  applied  to 
the  opposite  edge  of  the  cover-g. 

"The    '  :ons  and  tables,  with  alight  alterations,  are   saken  from 

Yanghan'3  Handbook  of  Chemical  Physiology  and  Pathology,  3rd  edition,  p.  323. 


284 


PHYSIOLOGICAL  CHEMISTRY. 


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Physiological  chemistry. 


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Fig.  11. 


Fi>.  10.  Sediment  in  acid  urine. — Calcium  oxalate,  uric  acid,  and  arnorphou- 
acid  urate*.    (HzuBavm  and Vocmbl.) 

Fi<,  'II.  Sediment  in  alkaline  urine.— Triple  and  amorphous  phohpaatee.  am- 
monium urate  and  bacteria.    'Nkuhaueis  and  Vogkx.j 


(2991 


Fig.  12. 


Fig.  13. 


Fig.  12.    Forms  of  uric  acid.    <Schotten.) 

Fio.  13.    Leucin  balls  on  the  left— ty  rosin  needles  on  the  right.    (NECBAtrERand 

VOGEL.) 


(301) 


Fig.  15. 


Fig.  14.    CyBtin.    (Neubauer  and  Vooel.) 
Fig.  15.    Cholesterin.    (Harley.) 


(303) 


Fig    iti. 


Fig.  11 


Fig.  16.    Forms  of  calcium  oxalate,     (v.  Jaksch.) 
Fie.  17.     Forms  of  calcium  carbonate.    (Vaughan.) 


(305) 


Fig.   18. 


Fig.   is). 


Fig.  18.    Ammonium  magnesium  phosphate  (Triple  phosphate),    (v.  Jaksch.) 

Fiu.  19.  Forms  of  casts,  a — granular  cast;  h— the  same  beset  with  renal  epithe- 
lium; c— the  same  with  colorless  blood  cells;  d— the  same  with  drops 
of  fat  and  fatty  crystals;  e — hyaline  cast  covered  with  red  blood  cells; 
/—the  same  with  renal  eithelium;  gr— cast  of  white  blood  cells,  (v. 
Jaksch.) 


307) 


Fig.  20. 


Fie.  20.    Cylindroids  from  the  urine  in  congested  kidney,    (v.  Jaksch.) 


(309) 


Fig.  21. 


<3   (spja?  "0     c 


Fie;.  21.    a — spermatozoa;    h — red  blood  cells,  some  crenated;    c — pus  corpuscles. 
(Schotten,  Neubauer  and  Vosel.) 


(311) 


Fig.  22. 


Fig.  23. 


Fig.  22.  a— micrococci;  f» — bacilli;  c— filaments  of  moulds;  d—  yeast-cells; 
e— micrococcus  ureie.    (v.  Jaksch.) 

Fitr.  23.  a— squamous  eqithelium;  b— epithelium  from  the  bladder;  c — epithe- 
lium from  the  kidneys;  d—  fatty  epithelium  from,  the  kidneys,  (v. 
Jaksch.) 


(313) 


Fig.  24 


Fig.  24.  Absorption  spectra  (after  Hammarsten):—  1.  A  solution  of  oxy-hserao- 
globin.  2.  A  solution  of  hemoglobin,  obtained  by  treating  oxy-haenio- 
globin  with  an  amraoniacal  ferro-tartrate  solution  (Stokes'  fluid). 
3  A  weak  alkaline  solution  of  methasmoglobin  4.  An  alkaline 
solution  of  hsematin.  5.  An  alkaline  solution  of  hsemochromogen. 
obtained  by  treating  an  alkaline  hseniatin  solution  with  Stokes'  fluid. 
6   An  acid  solution  of  urobilin. 


(315) 


List  of  Reagents.* 

Acid,  Acetic,  HC2H302.     Glacial. 

Acetic,  dilute.     Sp.  gr.  1.04;  about  30  per  cent.  acid. 
Hydrochloric,  HC1,  concentrated.     Sp.  gr.  1.19 — 1.20; 

about  38 — 39  per  cent.  acid. 
Hydrochloric,    dilute.       Sp.    gr.    1.10;    about   20   per 

cent.  acid. 
Hydrosulphuric,  H2S.     Used   either   in   the   form   of 

gas,  or  saturated  aqueous  solution. 
Iodic,  HI03;  1:20. 
Nitric,  fuming.     Sp.  gr.  about  1.50. 
Nitric,    HN03,    concentrated.       Sp.    gr.    1.42;    about 

69 — 70  per  cent.  acid. 
Nitric,  dilute.     Sp.  gr.  1.20;  about  32 — 35  per  cent. 

acid. 
Oxalic,  H2C204.2  H20;  1:  10. 
Oxalic,  standard  solution;  see  p.  239. 
Phosphomolybdic;  1:  20. 
Phosphotungstic;  1:  20. 

Picric,  C6H2(N03),OH;  saturated  solution;  about  1:100. 
Rosolic,  alcoholic  solution;  1:100. 
Sulphanilic;  see  p.  193. 

Sulphuric,  H2S04,  concentrated.     Sp.  gr.  1.84. 
Tannic,  1:100. 
Tannic,  Almen;  see  p.  270. 
Alcohol,  C2H5OH,  absolute.     Sp.  gr.  .79. 
Alcohol,  ordinary.     Sp.  gr.  about  .815;   about  95  per  cent. 
Ammoniacal  Silver  Nitrate;  see  p.  19  and  254. 
Ammonium  Carbonate,  (NH4)2C03;  1:4;  add  1  part  of  NH4OH, 

sp.  gr.  .96. 
Ammonium  Chloride,  NH4C1;  1:  8. 

Ammonium  Hydroxide,  NH4OH.     Sp.  gr.  .96;  about  10  per 
cent.  NH3. 

♦Unlcm*  otherwise  specified,  waler  in  to  be  used  an  a  nolvent. 


318  PHYSIOLOGICAL  CHEMISTRY. 

Ammonium  Molybdate,  (NH4)2Mo04.  Dissolve  15  parts  in 
100  parts  of  hot  water,  and  pour  into  100  parts  of 
HNOs  sp.  gr.  1.20. 

Ammonium  Oxalate,  (NH4)2C204. H20 ;  1:  24. 

Ammonium  Sulphate,  (NH4)2S04;  saturated  solution,  about 
80:100. 

Ammonium  Sulphide,  (NH4)2S;  saturate  NH4OH,  sp.  gr.  .96 
with  hydrogen  sulphide,  then  add  an  equal  vol- 
ume of  NH4OH,  sp.  gr.  .96. 

Barfoed's  Reagent;  see  p.  20. 

Barium  Chloride,  BaCl2.2  H20;  1:10. 

Barium  Chloride,  standard  solution;  see  p.  214. 

Barium  Hydrate,  Ba(OH),;  saturated  solution,  1:30. 

Barium  Nitrate,  Ba(N03)2;  1:15. 

Baryta  Mixture;  see  p.  251. 

Boas'  Reagent;  see  p.  69. 

Bromine  Water;  saturated  solution. 

Calcium  Chloride,  CaCl2;  1:  8. 

Calcium  Hydrate,  Ca(OH)2;  saturated  solution. 

Calcium  Hypochlorite,  CaCl2.Ca(OCl)2;  saturated  solution, 
1:  700. 

Chlorine  Water;  saturated  solution. 

Cleaning  Mixture.  Dissolve  80  parts  of  K2Cr207  in  350  c.  c. 
of  water,  and  then  add  cautiousl}?-  450  c.c.  of 
H2S04. 

Cochineal.  Dissolve  1  part  in  100  parts  of  25  per  cent, 
alcohol. 

Copper  Sulphate,  CuS04.5  H20;  1:8;  one-half  per  cent,  solu- 
tion, or  less,  for  biuret  test. 

Dimethyl  amido-azobenzol;  .5  per  cent,  alcoholic  solution. 

Ehrlich's  Reagent;  see  p.  193. 
Ether,  (C2H5),0.     Sp.  gr.  about  .725. 

Fehling's  Solution;  see  p.  247. 


tJST  OF  i:i;ai; knTO.  319 

Ferric  Alum,  K2SCvFe,.(S04);,  +  24  11,0;  1:  20. 

Ferric   Chloride,  FeCl3;  1:15;  almost  colorless  solution  for 

lactic  acid  test. 
Furfurol,  concentrated;  1:  50. 
Furfurol,  dilute;  1:1000. 

Guaiacum;  1:30;  alcoholic  solution.     To  be  used  fresh. 
Gi'mzburg's  Reagent;  see  p.  68. 

Iodine  Solution  (Lugol's  solution);  1  part  of   iodine,  2  parts 

of  potassium  iodide,  300  parts  of  water. 
Iodine  Tincture;  7  per  cent,  alcoholic  solution. 

Lead  Acetate,  Pb(C2Ha02)a.3  H20;  1:10. 

Lead  Acetate,  basic.  Dissolve  17  parts  of  Pb(C2H302)2  in 
hot  water,  add  10  parts  of  lead  oxide,  boil  for 
half  an  hour,  dilute  to  100  parts  and  filter. 

Litmus  Solution.  Digest  1  part  in  6  fjarts  of  water  and 
filter. 

Magnesia  Mixture;  see  p.  254. 

Magnesium    Sulphate,    MgS04.7H20;     saturated    solution, 

about  120  :  100. 
Mercuric  Chloride,  HgCl2;  1:16. 
Mercuric  Nitrate,  Hg(N03)2;  see  p.  251. 
Methyl  Violet;  .5  grams  in  1000.  c.c.  of  water. 
Millon's  Reagent;  see  p.  40. 

a-Naphthol;  alcoholic  solution,  15  :  100. 

Naphthylamin  Hydrochloride;  saturated  solution.     To  be 

used  fresh. 
Nylander's  Solution;  see  p.  20. 

Phenol  Phthalein;  alcoholic  solution,  1:1000. 
Potassium  Alum,  K:S04.A1,(S04)3  +  24  H20;  saturated  solu- 
tion. 
Potassium  Chromate,  K2Cr04;  1:10. 
Potassium  Dichromate,  K2Cr207;  1  :  10. 


320  PHYSIOLOGICAL    CHEMISTRY. 

Potassium  Ferricyanide,  K3Fe(CN)6;  1  :  12. 

Potassium  Perrocyanide,  K4Fe(CN)0.3  H20;  1  :  12. 

Potassium  Hydroxide,  KOH;  1  :  10. 

Potassium  Iodide,  KI;  1  :  20. 

Potassium  Mercuric  Iodide,  (Mayer's  Reagent);  see  p.  32. 

Potassium  Permanganate,  KMn04;  1  :  20. 

Potassium  Permanganate,  standard  solution;  see  p.  256. 

Potassium  Sulphide,  K2S;  see  p.  254. 

Potassium  Sulphocyanate,  KCNS;  1  :  12. 

Potassium  Sulphocyanate,  standard  solution;  see  p.  241. 

Schweizer's  Reagent;  see  p.  35. 

Silver  Nitrate,  AgN03;  1  :  20. 

Silver  Nitrate,  standard  solution;  see  p.  241. 

Sodium  Acetate,  NaC2H302.3  H20;  1  :  5. 

Sodium  Alizarin  Sulphonic  Acid;  see  p.  272. 

Sodium  Carbonate,  Na2C03;  1  :  5. 

Sodium  Carbonate,  indicator;  see  p.  251. 

Sodium  Chloride,  NaCl;   saturated  solution,  about  35  :  100. 

Sodium  Chloride;  1  :  10. 

Sodium  Hydroxide,  NaOH;  1  :  9. 

Sodium  Hydroxide,  for  Kjeldahl  distillation;  1  :  2. 

Sodium  Hydroxide,  standard  solution;  see  p.  239. 

Sodium  Hypobromite,  NaBrO;  see  p.  252. 

Sodium  Indigo  Sulphate,  (Indigo  Carmine);  1  :  20. 

Sodium  Nitrite,  NaN02;  1  :  200. 

Sodium  Nitroprusside;  1  :  20.     To  be  used  fresh. 

Sodium  Phosphate,  Na2HPO,.12  H,0;  1  :  10. 

Sodium  Sulphide,  Na2S;  see  p.  254. 

Starch  Paste;  see  p.  28. 

Stokes'  Solution;  see  p.  102. 

Tropaeolin  OO;  see  p.  69. 

Uffelmann's  Reagent;  see  p.  70. 
Uranium  Acetate;  see  p.  245. 

Zinc  Chloride,  ZnCL;  1  :  20. 


INDEX. 


Acetacetic  acid 179 

Acetic  acid 34,  61 

Aceton 176 

Acetonuria 176 

Achromic  point..    59 

Achroodextrin... 15,  28 

Acid  albumin 38,  46,  65 

"     calcium  phosphate 200 

"     fermentation 235 

"      sulphur 195 

Acidity,  estimation 239 

Acrolein 11 

Acrylic  aldehyde  11 

Adamkiewicz's  reaction 40 

Adenin  ... 153 

Adipocire 9 

Albumin 38,  42,  45,  183 

"        estimation 261,  263 

"        tests 39,  111,  184 

Albuminoids 38 

Albuminous  bodies 38 

Albuminuria 184 

Albumose...3S,  42,  45,  49,  65,  79, 

185,  211 

"        detection 50,186 

"        estimation 264 

tests 50 

Albumosuria 186 

Alimentary  glycosuria 17,  78 

Alkali  albuminate 38,  46 

Alkaline  hajmatin 104 

phosphates 198 

Alkalinity,  estimation 239 

Alkaloidal  reagents 44 

Alkapton 16,  19 

Alkaptonuria 170 

Allantoic  acid 152 

Allantoin 146,  150,  1 52 

A  I  latitude  acid 152 

Alloxan 145,  146,  149,  152 

Alloxanic  acid 152 

Alloxantin 145,  148,  152 

Alloxuric  bases 256 

bodies 250 

Almen's  guajac  test 105 

Amido  acids. 37,  126 

Amido-barbituric  acid... 152 

Ammonia 87,79,  136 

Ammoniacal  fermental  Ion 137 


Ammonium  carbamate 126 

"  carbonate 126 

purpurate,  see  Mur- 
exid. 

"  urate  calculi 213 

Ampho-pepton 66 

Amylo-dextrin 15 

Amyloid 33,  35,  38 

"        casts,  see  Waxy  casts. 

Amylopsin 77 

Amylum,  see  Starch. 

Animal-gum 181 

Animal-fat 7 

Animal  organisms 208 

Anti-albumose 66 

Anti-pepton 66,  79 

Anuria 226 

Arginin 37 

Asparaginic  acid.., 79 

Bacteria 57,  206,  299,  313 

Barbituric  acid 152 

Barfoed's  test 20 

Benzoic  acid 156,  161 

Benzopurpurin  test 69 

Benzoyl  chloride  test 133 

"    "     urea 133 

Bile 87,  193 

"     acids 88,  193 

"         "     tests 90 

"     pigments 88,  193 

"        tests 92 

"     stones 93 

'•         "      analysis 94 

Bilicyanin 89 

Bilifuscin 89 

Biliprasin 89 

Bilirubin 88,  211 

Biliverdin 88 

Biuret 134 

"       test 39,134 

Blood 97,  188 

"     casts 206 

"    coagulation 100 

"     corpuscles 97,  203,  277,  31 1 

"     examination 273 

"     plasma 99,  109 

"     plates 99 

"    serum 109 


INDEX. 


Blood  spectroscopic  examina- 
tion  102,  315 

Bloxam's  test '. 133 

Boas'  test 69 

Bottger's  test 20 

Bromine  reaction 81 

Butter 8 

"       fat 12 

Butyric  acid 8,  34,  61 

Cadaverin 172,  174 

Caffein 153 

Calcium-bilirubin 93 

Calcium  carbonate 210,  305 

"  "  calculi 213 

"        oxalate  ..175,  210,  299,  305 

"  "        calculi 213 

"        phosphate 199,  200 

"        sulphate 210 

Cancer  cell 204 

Cane  sugar 15,16,  22 

Caramel  18 

Carbamic  acid 127 

Carbamide,  see  Urea. 

Carbohydrates 15,  181 

"  absorption 78 

classification....  15 

Carbolic  urine 168 

Carbon  monoxide  haemoglobin 

104,  106 

Casein 38,  116 

Casts 184,  204,  307 

Cellulose 15,  32 

Cercomonas 208 

Chlorides 200 

"        estimation 241 

Cholesterin 

...88,90,93,  94,  180,  211,  303 

Cholesterin  calculi 93,  95,  214 

"  tests 95 

Cholic  acid 88 

Chrysophanic  acid 20 

Chyle 10,  99 

Chyluria 180 

Chymosin,  see  Rennin. 

Coagulation  point 45 

"  test.. 43 

Collagen 38,  79 

Collodium 34 

Colostrum  corpuscles 118 

Congo-red  test 69 

Colorimetric  method 274 

Conjugate  sulphates 195 

"        estimation..245 

Coprosterin 95 

Creatin 153 

Creatinin 153 

Cresol,  see  Phenol. 


Cyanuric  acid 134 

Cylindroids 206,  309 

Cystei'n 172 

Cystin 172,  210,  303 

"      calculi 174,  214 

Cystinuria 172 

Cystosin 192 

Czapek's  method 255 

Defibrinated  blood 100,  102 

Densimetric  method 261 

Deutero-albumose 53,  66 

Devoto's  method 52 

Dextran 15 

Dextrin 29 

Dextrose 15, 16,  17,  182 

"        estimation 247 

"        tests 17 

Diacetic  acid,  see  Acetacetic 
acid. 

Diaceturia 179 

Dialuramid 145,  152 

Dialuric  acid 145,  152 

Dialysis 48 

Diamines 174 

Diastase 25,  38,  56 

Diazo  reaction 193 

Di-methylamido-azobenzoltest  68 

Di-saccharides 15 

Distomum  haematobium 208 

Donne's  test 191 

Doremus'  method 253 

Drechsel's  reaction 145 

Dumas'  method 260 

Dynamite 14 

Dys-albumose 65 

Dys-pepton 64 

Earthy  phosphates 198 

Echinococci 208 

Egg  albumin , 38,  39 

"     globulin 38 

Ehrlich's  reaction 193 

Einhorn's  saccharimeter 250 

Elastin 38 

Empirical  solution 218 

Enzymes 38 

Epithelial  casts 205 

"  cells 202.  313 

Erythrodextrin 15,  28 

Esbach's  albuminometer 262 

Ethereal  sulphates 195 

Exudates 99 

Factor 221 

Fats 7,  88,  118,  180 

"    absorption ....9,  77 

"    emulsification....8,  9,  13,  76,  8Q 


INDfiX. 


328 


Fats  preparation 10,  79 

•■    saponification 12 

Fat-splitting       ferment,      see 
Steapsin. 

Fatty  acid  12,  27 

••      casts 206 

Fehling's  solution 247 

'•   *"      test 19 

Fermentation  test 21 

Ferrocyanide  test 43,  185 

Fibrin 38,  100,  113,  188 

"      ferment 100,  114 

Fibrinogen 38,  99,  113,  183 

Filaria  sanguinis 208 

Fixed  alkali 236 

Fleischl's  haemometer 274 

Folin's  method 256 

Formic  acid 24 

Fructose,  see  Laevulose. 
Furfurol  test 91,  133 

Galactite 22 

Galactose -17,  22,  24 

Gastric  juice 61 

"     analysis 74,  272 

Gelatin 38,  54 

"        pepton 79 

Gerhardt's  test 179 

Globulin 38,  42,  45,  47,  183 

"        detection 48 

"        estimation 261,  263 

Glucose,  see  Dextrose. 

Glucosin 26 

Glycerin 7,  13,  76 

"        phosphoric  acid 198 

Glycocholic  acid 88 

Glycocoll 88,  156,  160 

Glycogen 15,  29 

(Jlyco-proteid 38 

(Uycose 15 

(Glycosuria 182 

(Hycuronic  acid 16,  157,  183 

(ilyoxyl-diureid,  see  Allantoin. 
<4lyoxyl    urea,   see   Allanturic 
acid. 

Gmelin's  test 92 

Oonococcus 207 

Granular  casts 205 

Grape  Bugar,  see  Dextrose. 

(iuajac  test 105,  120,  191 

Guanin 153 

Gun  cotton 34 

Gunning's  test 178 

Giinzburg's  test 68 

Baematin 42,  89,  107 

I  [aematoidin 89 

Hamatopor  phy  r  i  m 1 04 .  1 89 


Hematuria 188 

Hamiin 107 

"        crystal  test 98,  107 

Ffemochromogen 103,  107 

Haemoglobin 38,  42,  189 

'•  estimation 274 

Hemoglobinuria 189 

Hamrnarsten's  method 2(33 

Haycraft*s  method 255 

Heintz's  method 254 

Heller's  test 42,  108 

Hemi-albumose 00 

Hemi-pepton 66,  79 

Hetero-albumose 65,  180 

He  teroxanthin 1  o'.'> 

Hexose 15,  10 

Hippuric  acid 156,  209 

"     tests 160 

Histidin 37 

Histon 38,  187 

Histozy  m 161 

Hoffmann's  reaction 85 

Hofmeister's  method 51 

Homogentisinic  acid 170,  183 

Hopkin's  method 256 

Hoppe-Seyler's  test 91,  100 

Hiifner's  method 252 

Humin  substances 17,  24 

Huppert's  reaction 92 

Hyaline  casts 204 

Hydantoic  acid 152 

Hydrobilirubin 89 

Hydrochloric  acid 61,  62 

"  "    estimation  ..272 

"  "    tests 68 

"                "    loosely  com- 
bined  61,  62 

Hydrogen  sulphide ....196 

Hydroquinon 171 

Hydrothionuria 197 

Hyper-acidity 63 

Hypo-acidity 63 

Hy posulphurous  ac id 198 

Hy  pox  anthin 1 53 

Indican,  see  Indoxyl. 

Indigo 19,  162,  211 

"      blue 19,  104 

"      calculi 214 

"      white 19 

Indigogen,  see  Indoxyl. 

Indol 102.  104 

Indoxyl  162 

red 103 

"        test 164 

Inosite 10 

lnulin 22,  20 

Invert  sugar 22 


324 


INDEX. 


Invertin 23,  38,  78 

Iodine  test 92 

Iodoform  test 178 

Iso-maltose 15,  26,  28 

Jaffe's  test 164 

Jolles'  test 93 

Kephir 25 

Keratin 38 

Kjeldahl  method 258 

Kumyss 25 

Lact-albumin 38,  117 

Lactic  acid 25,  61,  116 

"        "      test 70 

Lacto-caramel 24 

Lacto-globulin 38,  117 

Lactose 16,  24,  118 

Lactosozon 25 

La^vulin 26 

Laevulinic  acid 17,  18,  24 

Laevulose 15,  16,  22 

Laky  blood 98,  106 

Lard 7,  12 

Lead  plaster 13 

Lecith-albumin 235 

Lecithin 88 

Legal's  test 178 

Leucin 37,  66,  79,  82,  175,  301 

"       tests 83,  86 

Leucocytes 98,  203 

Lieben's  test 178 

Liebermann's  reaction 40 

"       cholesterol  reaction  95 

Liebig's  method 251 

Lipaciduria 180 

Lipuria 180 

List  of  reagents 317 

Lithemia 141 

Ludwig's  method 254 

Lymph 10,  99,  125 

Lymphoid  cells 98 

Lysidin 143 

Lysin 37,  79 

Magnesium    ammonium   phos- 
phate  200,  299,  307 

Malonyl  urea,   see  Barbituric 
acid. 

Maltodextrin 28 

Maltose 15,  16,  25,  28 

Maltosozon 26 

Mannite 15,  16 

Margaric  acid 10 

Margarine 10 

Marsh  gas 34 

Melting  point,  determination..l31 


Mesoxalic  acid 149 

Mesoxalyl  urea,  see  Alloxan. 

Methaemoglobin 103,  190 

Methyl  violet  test 69 

Micrococcus  ureas 207,  313 

Milk 116 

"     analysis 267 

"     composition 116,  271 

"     fat 8,  118 

"     serum 116 

"    sugar,  see  Lactose. 

"     tests..... 119 

Millon's  reaction 39 

Mitscherlich  polarimeter 35 

Mohr's  method 242 

Molisch's  reaction 17 

Mono-saccharides 15,  16 

Moore-Heller  test '  18 

Morner-Sjoqvist  method 253 

Morner's  test 179 

Moulds 208,  313 

Mucic  acid 22,  25 

Mucin 38,  57,  58,  87,  183,  187 

Mucoid 38 

Mucous  corpuscles 203 

Murexid 145,  152 

"        test 145 

Muscle  albumin 38 

Myosin 38 

Neubauer's  method 155,  233 

Neutral  calcium  phosphate 199 

"         sulphur 195,  197 

Nitric  acid  test 43,  111,  185 

Nitro-cellulose 34 

Nitro-glycerin 14 

Nitroprusside  test 178 

Nitrogen,  estimation 258 

Nitrous  acid 58 

Normal  solution 220 

Nuclein 38,  64 

"       bases,     see     Xanthin 
bases. 

Nucleinic  acid 192 

Nucleo-albumin 38,  42,  87,  187 

Nucleo-histon 38,  191 

Nucleo-proteid 64 

Nylander's  test.. 20 

Oleic  acid 12 

Olein 7 

Oleomargarine 10 

Oliguria ' 226 

Organized  sediment 202 

Ornithin 158 

Ornithuric  acid 158 

Osazon 21 

Oxalate  plasma 100 


INDEX. 


325 


Oxalic  acid 28,  146,  175 

Oxaluria 175 

Oxaluric  acid 140,  152 

Oxalyl    urea,    see    Parabanic 
acid. 

Oxidized  sulphur 195 

Oxy-butyric  acid 179 

Oxy-haimog'lobin 102 

( )xyuris  vermicularis 208 

Palmitic  acid 10 

Palmitin 7 

Pancreatic  secretion 76 

Papayotin 38,  64 

Parabanic  acid 140,  152 

Para-casein 38,  117,  120 

Para-globulin,  see  Serum  glo- 
bulin. 

Para-nuclein 17 

Paraxanthin 531 

Parchment 33,  35 

Pavy's  solution 20 

Pentite 10 

Pentosane 15,  10 

Pentose 15,  16,  181 

Pentosuria 181 

Penzoldt's  test 178 

Pepsin 38,  61,  121 

"       test 74 

Pepsin-hydrochloric  acid 02 

Peptic  digestion 71 

Pepton..38,  42,  45,  51,  00,  79,  99,  180 

"        detection 51 

Pettenkofer's  test 90 

Phenol 102,  166,  169 

Phenyl-glucosozon 21 

Phenyl-hydrazine  test 21 

Phloro-glucin  vanillin  test 68 

Phosphates 210,  299 

Phosphatic  calculi 213 

1  'hosphoric  acid 198 

"  "     estimation. ...245 

Phytosterin 95 

Phospho-sarkinic  acid 07 

Pialyn,  see  Steapsin, 

Piperazin 143 

Piria's  reaction 85 

Plant  albumin 38 

"      globulin 38 

Pohl's  method 263 

Poly-saccharides 15 

Polyuria 184,  220 

Potassium  phenol  sulphate 168 

Primary  albumose 65 

calculi 212 

I  'n>1ri<ls 38 

tests 39 

Proteins 37 


Proteins  classification 38 

Proteinochrom 81 

Proteinochromogen 79,  si 

Proteose 00 

Prothrombin 101,  114 

Proto-albumose 65 

Pseudo-casts  200 

Pseudo-nuclein 04,  117 

Ptyalin 50,  59 

Ptyalism 55 

Purpuric  acid 145,  152 

Pus 105,  190,203,  311 

"     casts 200 

Putrescin 178,  174 

Pyrocatechin 25,  169 

Pyroxylin 34 

Pyuria 190 

Quantitative  analysis 217 

of  blood..273 
"                    "of     gas- 
tric juice 272 

"  analysis  of  milk  ..207 

"  "of  urine.  225 

Reduced  haematin 103 

"        haemoglobin 102 

Rennin 38,  01,  07 

Rhamnose 15 

Roberts-Stolnikoff  method 202 

Rosenbach's  test 92 

Saccharic  acid     25,  28 

Saccharide 15 

Saccharose,  see  Cane  sugar. 

Saliva 55 

Salivary  calculi 50 

Sarkinic  acid 67 

Seherer's  method 261 

test 84,  85 

Schweizer's  reagent 34 

Secondary  albumose 00 

"  calculi 212 

Serum  albumin 38,  47,  99 

"       globulin 38,  47,  99,  110 

Skatol 162,  105 

Skatoxyl   105 

Skeltal  substances 38 

Soap 13,  76,  88 

Soleil-Ventzke  saccharimeter.  36 

Soluble   starch 27,  28 

Specific  gravity,  determination 

232,  207,  273 

Spermatozoa 204,  .'ill 

Standard  solutions 218 

Starch 15,  20 

•'      granules 27 

"       iodide 28 


326 


INDEX. 


Starch  paste 28 

"        sugar,  see  Dextrose. 

Steapsin y,  38,  76,  80 

Stearic  acid 10 

Stearin 7 

Stercobilin ■. 8y 

Stercorin y5 

Stokes'  reagent 102 

Stomach   contents,  examina- 
tion    74 

Stroma 98 

Sucrose,  see  Cane  sugar. 

Suet 7 

Sulphocyanate 58,  194 

Sulphur ....194 

Sulphur-methaemoglobin 106 

Sulphuric  acid 197 

"  "     estimation 244 

Syntonin,  see  Acid  albumin. 

Table  of  atomic  weights 216 

Tallow 7,  12 

Tartar 56 

Tartaric  acid 28 

Tartronyl  urea,    see   Dialuric 
acid. 

Taurin 88,  195 

Taurocholic  acid 88 

Thein 153 

Theobromin 153 

Thiosulphuric  acid 198 

Thrombosin 101 

Thymic  acid 192 

Thymin 192 

Tissue  debris 204 

Toepfer's  method 272 

Transudates 99 

Trichomonas  vaginalis 208 

Triple  phosphate,  see  Magne- 
sium ammonium  phosphate. 

Trommer's  test 19 

Tropaeolin  OO  test 69 

Trypsin 38,  78 

Trypsinogen 78 

Tryptophan 81 

Tubercle  bacillus 7,  207 

Tunicin 32 

Tyrosin...37,  66,  79,  84,  175,  210,  30  L 
"       tests 85,  86 

Uffelmann's  test 70 

Unknowns 75,  277,  280 

Unorganized  sediment 209 

Unoxidized  sulphur 195 

Uraemia 128 

Uramil,  see  Dialuramid. 

Urase 38 


Urates 209,  299 

Urea 88,  125 

"     detection 136 

"     estimation 251 

"     nitrate 132 

"     oxalate 132 

"     preparation 129 

"     properties 131 

"     tests 133 

Uric  acid 

125, 138, 152, 153,  209,299,301 

"       "    calculi 213 

',       "     estimation 254 

"       "     preparation 142 

"       "     properties 143 

"       "     tests 144 

Urinary  calculi 211 

"    '        "       analysis 214 

sediment 201,283 

Urine 123 

"     composition 265,266 

"     examination 283 

"     quantity 225 

"     reaction 234 

"     specific  gravity 230,  253 

"     total  solids 233 

Urobilin 89 

Urocy  anin 1 65 

Uroglaucin 165 

Uroleucinic  acid 170,  183 

Urohaematin 165 

Urophaein 124 

Urorosein 165 

Urorubin 165 

Urostealith 214 

Varrentrapp-Will  method 260 

Vegetable  oils 7 

"  organisms 206 

Vitali's  guajac  test 191 

Vitellin 38 

Volatile  alkali 138,  236 

Volhard's  method 241 

Waxy  casts 205 

Whey H6 

"     proteid 117 

Winternitz  test 43 

Wood-silk 34 

Xanthin 139,  153,  211 

"       bases 139,  153 

"       calculi 214 

Xantho-creatinin 155 

Xantho-proteic  reaction 40 

Yeast  cells 208,  303 


f\  LIST  OF  BOOKS 

PUBLISHED  BY 

GEO.      "v^T^L-IHIIR 

Publisher  and  Bookseller  to  the  University  hf  Michigan, 
Axn  Arbor. 


Any  book  in  this  list  well   be  sent,  carriage  free,  to  any  address  in  the 
world  on  receipt  of  price  named. 

BOWEN. — A  Teachers'  Cause  in  Physical  Training.  By  Wilbur  P. 
Bowen,  Director  of  Physical  Training,  Michigan  State  Normal  Col- 
lege.    In  Press. 

CHEEVER. — Select  Methods  in  Inorganic  Quantitative  Analysis.  By 
Byron  W.  Cheever,  A.M.,  M.D.,  late  Acting  Professor  of  Metal- 
lurgy in  the  University  of  Michigan.  Revised  and  enlarged  by  Frank 
Clemes  Smith,  Professor  of  Geology,  Mining  and  Metallurgy  in  the 
State  School  of  Mines,  Rapid  City,  S.  D.  Parts  I.  and  II.  Third 
edition.     i2nio.     $1.75. 

The  first  part  of  this  book,  as  indicated  by  the  title,  consists  of  Laboratory  Notes 
for  a  Beginner's  Course  in  Quantitative  Analysis.  It  considers  the  subjects  of 
Gravimetric  and  Volumetric  Analysis,  for  beginners,  by  means  of  the  chemical 
analysis  of  a  set  of  substances,  properly  numbered,  in  each  case  giving  the  methods 
to  be  followed  in  such  analysis;  also  the  methods  for  calculating  and  preparing 
volumetric  standard  solutions,  generally  following  the  course  offered  by  Professor 
Cheever  to  his  students.  It  also  considers  the  methods  for  the  determination  of  the 
specific  gravities  of  various  liquids  and  solids. 

Although  a  number  of  the  analyses  contained  in  Part  I.  may  be  of  only  approxi- 
mate accuracy,  and  of  small  commercial  value,  such  are  yet  included  with  a  special 
purpose,  to  wit:— that  they  may  supply  the  student  with  a  wider  range  of  work  and  a 
greater  diversity  of  chemical  manipulation.  This  was  Professor  Cheever's  idea, 
and  it  is  certainly  a  good  one,  especially  since,  in  most  cases,  the  work  of  the  begin- 
ner simply  serves  to  emphasize  the  necessity  of  careful  scrutiny  of  details  and 
methods  for  practical  work  in  the  future. 

Part  I.  is  offered,  then,  for  the  use  of  schools  and  colleges,  and  it  is  intended  to 
supply  a  source  of  elementary  information  upon  the  subject  of  Quantitative  Chemi- 
cal Analysis  rarely  offered  in  such  form  in  works  upon  that  sublet. — Preface. 

The  author  was  for  many  years  Professor  of  Metallurgy  in  the  Uuiversity  of 
Michigan,  and  the  methods  here  presented  are  those  mostly  offered  by  him  to  his 
students.  As  a  beginner's  book  in  quantitative  analysis,  it  will  be  found  eminently 
practical,  and  it  can  be  honestly  recommende*'  to  the  student  who  desires  a  source 
of  elementary  information  upon  this  branch  of  applied  science  The  book  is  divided 
into  two  parts,  the  first  consisting  of  laboratory  notes  for  beginners.  The  subjects 
of  gravimetric  and  volumetric  analysis  are  considered  by  means  of  the  chemical 
analysis  of  a  set  of  substances,  propel  ly  numbered,  in  each  case  giving  the  methods 
to  be  followed  in  such  analysis,  and  also  the  methods  of  calculating  and  preparing 
volumetric  standard  solutions,  etc.  Methods  for  the  determination  of  specific 
gr.i  /i ties  of  various  liquids  and  solids  are  also  considered. 

Part   II.  contains  a  number  •     thods  in  inorganic  quantitative  analysis, 

such  as  the  analysis  of  limestone,  iron  ores,  manganese  ores,  steel,  the  analysis  of 


coal,  water,  mineral  phosphates,  smelling  ores,  lead  slags,  copper,  arsenic,  bismut  h, 
etc.     A  chapter  on  reagents  concludes  the  work. — Phar  maceuiical  E)a. 

DEWEY. —  The  Study  of  Ethics.  A  Syllabus.  By  jonn  Dewey,  Pro- 
fessor of  Philosophy  in  the  University  of  Chicago.  Octavo.  144 
pages.     Cloth,  $1.25. 

D'OOGE. — Helps  to  the  Study  of  Classical  Mythology;  for  the  Lower 
Grades  and  Secondary  Schools.  By  B.  L.  D'Ooge,  Professor  in  the 
Michigan  State  Normal  College.    12  mo.    1S0  pages.    Cloth.    45  cents. 

A  bibliography  based  on  practical  experience.  The  author  is  a  professor  in  the 
Michigan  State  Normal  College.  As  the  myths  of  all  nations  manifest  themselves 
first  in  religion,  secondly  in  art,  and  third  in  literature,  these  reading  references  are 
grouped  in  the  above  classes.  One  section  is  devoted  to  the  study  of  mythology  in 
the  grades,  and  an  introductory  chapter  gives  hints  for  teaching  the  subject  in  the 
lower  grades.  The  books  suggested  in  the  body  of  the  work  are  given  in  one  alpha- 
bet at  the  end,  with  publishers  and  prices;  there  are  also  blank  pages  for  additional 
references,  and  a  good  general  index. — Publishers  Weekly, 

DOW. — Brief  Outlines  in  European  History.  A  Syllabus  for  the  Use  of 
Students  in  History,  Course  I.,  in  the  University  of  Michigan.  By 
Earl  Wilbur  Dow.     41  pages.     Pamphlet,  35  cents. 

DOW. — Brief  Outlines  in  European  History.  A  Syllabus  for  the  Use  of 
Students  in  History,  Course  II.,  in  the  University  of  Michigan.  By 
Earl  Wilbur  Dow.     47  pages.     Pamphlet,  35  cents. 

DZIOBEK. — Mathematical  Theories  of  Planetary  Motions.  By  Dr. 
Otto  Dziobek,  Privatdocent  in  the  Royal  Technical  High  School  of 
Berlin,  Charlottenburg.  Translated  by  Mark  W.  Harrington,  for- 
merly Chief  of  the  United  States  Weather  Bureau,  and  Professor  of 
Astronomy  and  Director  of  the  Observatory  at  the  the  University  of 
Michigan,  President  of  the  University  of  Washington,  and  Wm.  J. 
Hussey,  Assistant  Professor  of  Astronomy  in  the  Leland  Stanford, 
Jr.  University.     8vo.     294  pages.     $3.50. 

The  determination  of  the  motions  of  the  heavenly  bodies  is  an  important  problem 
in  and  for  itself,  and  also  on  account  of  the  influence  it  has  exerted  on  the  develop- 
ment of  mathematics.  It  has  engaged  the  attention  of  the  greatest  mathematicians, 
and,  in  the  course  of  their  not  altogether  successful  attempts  to  solve  it,  they  have 
displayed  unsurpassed  ingenuity.  The  methods  devised  by  them  have  proved  use- 
ful, not  only  in  this  problem,  but  have  also  largely  determined  the  course  of  advance 
in  other  branches  of  mathematics.  Analytical  mechanics,  beginning  with  Newton, 
and  receiving  a  finished  clearness  from  Lagrange,  is  especially  indebted  to  this 
problem,  and  in  turn,  analytical  mechanics  has  been  so  suggestive  in  method  as  to 
determine  largely  both  the  direction  and  rapidity  of  the  advancement  of  mathemat- 
ical scienoe. 

Hence,  when  it  is  desired  to  illustrate  the  abstract  theories  of  analytical  mechan- 
ics, the  profundity  of  the  mathematics  of  the  problem  of  the  motions  of  the 
heavenly  bodies,  its  powerful  influence  on  the  historical  development  of  this 
science,  and  finally  the  dignity  of  its  object,  all  point  to  it  as  most  suitable  for  this 
purpose. 

This  work  is  intended  not  merely  as  an  introduction  to  the  special  study  of 
astronomy,  but  rather  for  the  student  of  mathematics  who  desires  an  insight  into  the 
creations  of  his  masters  in  this  field.  The  lack  of  a  text-book,  giving,  within  mode- 
rate limits  and  in  a  strictly  scientific  manner,  the  principles  of  mathematical  astron- 
omy in  their  present  remarkably  simple  and  lucid  form,  is  undoubtedly  the  reason 
why  so  many  mathematicians  extend  their  knowledge  of  the  solar  system  but  little 
beyond  Kepler's  law.  The  author  has  endeavored  to  meet  this  need,  and  at  the 
same  time  to  produce  a  book  which  shall  be  so  near  the  present  state  of  the  science 
as  to  include  recent  investigations  and  to  indicate  unsettled  questions. 

FORD.—  The  Cranial  Nerves.  12  pairs.  By  C.  L.  Ford,  M.D.,  late 
Professor  of  Anatomy  and  Physiology  in  University  of  Michigan. 
Chart,  25  cents. 


FORD.— C/assiJica/ion  of  the  Most  Important  Muscles  of  the  Human 
Body,  With  Qrigin  Insertion,  Nervous  Supply  and  Principal  Action 
of  Each.  By  C.  L.  Ford,  M.D.,  late  Professor  of  Anatomy  and 
Physiology  in  the  University  of  Michigan.      Chart,  50  cents. 

FRANCOIS.  —Les  Aventures  Du  Dernier  Abencerage  Par  Chateaubri- 
and, Edited  with  Notes  and  Vocabulary.  By  Victor  E.  Francois, 
Instructor  in  French  in  the  University  of  Michigan.  Pamphlet,  35 
cents. 

GRAY. — Outline  of  Anatomy.     A  Guide  to  the  Dissection  of  the  Human 
Body,  Based  on  Gray's  Anatomy.      54  pages.     Boards,  60  cents. 
The  objects  of  the  outline  are  to  inform  the  students  what  structures  are  found 
in  each  region  and  where  the  description  of  each  structure  is  found  in  Gray's  Ana- 
tomy.— Thirteenth  edition,  dated  1897. 

GREENE.  —The  Action  of  Materials   Under  Stress,  or  Structural  Me- 
chanics.      With    examples    and    problems.      By  Charles    E.  Greene, 
A.M.,  M.E.,   Professor  of    Civil    Engineering  in  the  University  of 
Michigan.     Consulting  Engineer.      Octavo.     Cloth,  $3.00. 
Contents.— Action  of  a  Piece  under   Direct  Force.     Materials.     Beams.     Tor- 
sion.    Moments  of  Inertia.     Flexure  and  Deflection  of  Simple  Beams.     Restrained 
Beams:  Continuous  Beams.      Pieces  under  Tension.      Compression  Pieces-— Col- 
umns, Pests   and    Struts.      Safe   Working   Stresses.      Internal   Stress:    Change   of 
Form.      Rivets:  Pins.      Envelopes:    Boilers,  Pipes,  Dome.      Plate  Girder.      Earth 
Pressure:  Retaining  Wall :  Springs:  Plates.     Details  in  Wood  and  Iron. 

HERDMAN-NAGLER.—  A  laboratory  Manual  of  Electrotherapeutics. 
By  William  James  Herdman,  Ph.B.,  M.D.,  Professor  of  Diseases  of 
the  Nervous  System  and  Electrotherapeutics,  University  of  Michigan, 
and  Frank  W.  Nagler,  B.S.,  Instructor  in  Electrotherapeutics,  Uni- 
versity of  Michigan.  Octavo.  Cloth.  163  pages.  55  illustrations. 
$1.50. 

It  has  been  our  experience  that  the  knowledge  required  by  the  student  of  medi- 
cine concerning  electricity  and  its  relation  to  animal  economy  is  best  acquired  hy 
the  laboratory  method.     By  that  method  of  instruction  each  principle  is  impressed 
upon  the  mind  through  several  separate  paths  of  the  sense  perception  and  a  manual 
xt^rlty  1S  aC(Iuired  winch  is  essential  to  success  in  the  therapeutic  applications. 

This  has  been  the  plan  adopted  for  teaching  electrotherapeutics  at  the  Univer- 
sity of  Michigan.  Every  form  of  electric  modality  that  has  any  distinctive  physio- 
logical or  therapeutical  effect  is  studied  in  the  laboratory  as  to  its  methods  of  gen- 
eration, control  and  application  to  the  pattent.  We  believe  this  to  be  the  only 
practicable  way  for  imparting  the  kind  of  instruction  required  for  the  practice  of 
electrotherapeutics,  but  in  our  attempt  to  develop  a  naturally  progressive  and  at  the 
same  tune  complete  and  consistent  course  of  laboratory  instruction  we  have  found  it 
a  thing  of  slow  growth. 

This  laboratory  manual  is  the  final  result  of  our  various  trials  and  experiences, 
and  while  we  do  not  claim  for  it  either  perfection  in  the  arrangement  of  matter  or 
completeness  in  detail,  we  feel  that  the  time  has  come  for  putting  our  plans  in  a  form 
that  will  permit  for  it  a  wider  usefulness  as  well  as  gain  for  it  in  the  intelligent  criticism 
of  the  experienced  workers  to  the  held  which  it  seeks  to  cultivate.—  From  Preface. 

HOWELL. — Directions  for  laboratory  Work  in  Physiology  for  the  Use 
of  Medical  Classes.  By  W.  H.  Howell,  Ph.D.,  M.D.,  Professor  of 
Physiology  and  Histology.      Pamphlet.      62  pages.     65  cents. 

HUBER.  —Directions  for  Work  in  the  Histological  Laboratory.  By  G. 
Carl  Huber,  M.D.,  Assistant  Professor  of  Histology  and  Embry- 
ology, University  of  Michigan.  Second,edition,  revised  and  enlarged. 
Octavo.      191  pages.     Cloth,  $1.50. 

rclassi      in  medical  schools  and  elsewhere  where  it  is  desired  to 
furnish  the  class  with  material  alien  !  foi    the  demonstration  of  structure 

rather  than  to  give  tn8iructioq.iq  ihe  technique  of  the  laboratory      Provision  for  the 


latter  is  made,  however,  by  the  addition  of  a  section  of  about  50  pages  on  the  meth- 
ods for  laboratory  work.  This  section  includes  methods  of  macerating,  hardening 
and  fixing,  decalcifying,  impregnation,  injecting,  embedding,  staining,  and  methods 
for  preparing  and  staining  blood  preparations.  The  last  is  accompanied  by  an  ex- 
cellent plate  of  blood  elements.  The  selection  of  methods  has  in  the  main  been 
judicious.  The  expositions  are  both  clear  and  concise. — Journal  of  Comparative 
Neurology. 

In  this  little  book  Dr.  Huber  has  given  us  a  model  manual  of  microscopical  tech- 
nique in  the  laboratory  study  of  histology.  The  subject  matter  is  divided  into  con- 
venient chapters,  commencing  with  the  cell  and  cell  division  (karyokinesis)  in  plant 
and  animal  life,  and  gradually  developing,  by  easy  stages,  the  most  complex  tissues 
of  the  animal  and  vegetable  organism.  Between  each  lesson  blank  pages  are  inter- 
leaved, to  be  used  by  the  student  for  drawing  the  objects  seen  by  him  with  a  pencil 
or  crayon — a  most  excellent  plan  as  nothing  fixes  the  appearance  and  characteristics 
of  objects  more  firmly  on  the  mind  ihan  drawing  them,  either  free-hand  or  with  a 
camera  lucida  (the  former  being  preferable,  as  it  educates  the  hand  and  eye).  With 
each  subject  is  given  the  source  and  origin,  the  best  methods  for  obtaining  and  pre- 
paring it,  and  attention  is  called  to  the  most  noteworthy  or  characteristic  points  for 
examination. 

The  second  part  of  the  book  is  devoted  to  methods  for  laboratory  work:  soften 
ing,  hardening,  decalcification,  etc.,  of  the  matter  in  gross;  embedding,  sectioning, 
staining  and  mounting,  eic.  The  best  stains,  with  methods  of  preparing  the  same, 
and,  in  short,  a  general  formulary  for  the  various  reagents,  etc.,  concludes  the  work, 
which  is  intended,  as  stated,  as  an  aide  meinoire  supplementary  to  a  course  of  lec- 
tures on  histology. 

We  congratulate  Dr.  Huber  on  the  skill  with  which  he  has  developed  the  idea, 
and  the  didactic  methods  whioh  he  has  employed.  Such  a  book  cannot  but  prove  a 
great  help  to  both  student  and  teacher,  and  it  should  be  more  widely  known. — St. 
Louis  Medical  and  Surgeon's  Journal. 

JOHNSON. —  Elements  of  the  Law  of  Negotiable  Contracts.  By  E.  F. 
Johnson,  B.S.,  LL.M.,  Professor  of  Law  in  the  Department  of  Law 
of  the  University  of  Michigan.  Svo.,  735  pages.  Full  law  sheep 
binding.     $3.75. 

Several  years  of  experience  as  an  instructor  has  taught  the  author  that  the  best 
method  of  impressing  a  principle  upon  the  mind  of  the  student  is  to  show  him  a  prac- 
tical application  of  it.  To  remember  abstract  propositions,  without  knowing  their 
application,  is  indeed-difficult  for  the  average  student.  But  when  the  primary  prin- 
ciple is  once  associated  in  his  mind  with  particular  facts  illustrating  its  applica- 
tion, it  is  more  easily  retained  and  more  rapidly  applied  to  analo  ous  cases. 

ft  is  deemed  advisable  that  the  student  in  the  law  sh  iuld  be  required,  during  his 
course,  to  master  in  connection  with  each  general  branch  of  the  law,  a  few  well-se- 
lected cases  which  are  illustrative  of  the  philosophy  of  that  subject.  To  require  each 
student  to  do  this  in  the  larger  law  schools  has  been  found  to  be  impracticable,  ow- 
ing to  a  lack  of  a  sufficient  number  of  copies  of  individual  cases.  The  only  solution 
of  this  difficulty  seems  to  be  to  place  in  the  hands  of  each  student  a  volume  contain- 
ing tiie  desired  cases.  In  the  table  of  cases  will  be  found  many  leading  cases  printed 
in  black  type.— From  Preface. 

LEVI-FRANCOIS- — A  French  Reader  for  Beginners,  with  Notes  ana 
Vocabulary.  By  Moritz  Levi,  Assistant  Prof essor  of  French,  Univer- 
sity of  Michigan,  and  Victor  E.  Fiancois,  Instructor  in  French,  Uni- 
versity of  Michigan.      12  mo.      261  pages.      $1.00. 

This  reader  differs  from  its  numerous  predecessors  in  several  respects.  First, 
being  aware  that  students  and  teachers  in  the  French  as  well  as  in  the  German  de- 
partm'  nts  of  high  schools  and  colleges  are  becoming  tired  of  translating  over  and 
over  again  the  same. old  fairy  tales,  the  editors  have  avoided  them  and  selected  some 
interesting  and  easy  short  stories.  They  have  also  suppressed  the  poetic  selections 
which  are  never  translated  in  the  class  room.  Finally,  they  have  exercised  the  great- 
est care  in  the  gradation  of  the  passages  chosen  and  in  the  preparation  of  the  vocab- 
ulary, every  French  word  being  followed  not  only  by  its  primitive  or  ordinary  mean- 
ing, but  also  by  the  different  English  equivalents  which  the  text  requires.  After 
careful  examination,  we  consider  this  reader  as  one  of  the  best  on  the  American 
market.  , 

LYMAN-HALL-GODDARD.-^/^ra.  By  Elmer  A.  Lyman,  A.B., 
Edwin  C.  Goddard,  Ph.B.,  and  Arthur  G.  Hall,  B.S.,  Instructor 
in  Mathematics,  University  of  Michigan.  Octavo,  75  pages.  Cloth, 
90  cents. 


MATTHEWS. — Syllabus  of  Lectures  on  Pharmacology  and  Therapeu- 
tics in  the  University  of  Michigan.  Arranged  Especially  for  the 
Use  of  the  Classes  Taking  the  Work  in  Pharmacology  and  Thera- 
peutics at  the  University  of  Michigan.  By  S.  A.  Matthews,  M.I)., 
Assistant  in  Pharmacy  and  Thorapeutice,  University  of  Michigan. 
i2mo.      114  pages.      $1.00. 

MEADER.  —  Chronological  Outline  of  Roman  Literature.  By  C.  L. 
Meader,  A.B.,  Instructor  in  Latin  in  University  of  Michigan. 
Chart,  25  cents. 

MICHIGAN  BOOK.—  The  U.  of  M.  Book.  A  Record  of  Student  Life 
and  Student  Organizations  in  the  University  of  Michigan.  Articles 
contributed  by  members  of  the  Faculty  and  by  prominent   Alumni. 

$1.50. 

MONTGOMERY-SMITH.—  Laboratory  Manual  of  Elementary  Chem- 
istry.    By  Jabez  Montgomery,   Ph.D.,  Professor  of  Natural  Science, 
Ann     Arbor    High    School,    and    Roy   B.    Smith,    Assistant     Profes- 
sor in  Chemical  Laboratory,  Ann  Arbor  High  School.      12  mo.      150 
pages.     Cloth,  $1.00. 
This  Work  is  intended  as  a  laboratory  guide  to  be  used  in  connection  with  a  good 
text-book  or  course  of  lectures,  and  in  its  arrangement  and  scope  it  is  based  upon 
the  practical  experience  of  two   instructors   in  the  Ann  Arbor    High  School.     It  i« 
therefore  restricted  to  s"ch  work  as  may  be  done  by  the  average  high  school  pupil. 
The  experiments  which  are  dii  ected  are  given  more  to  enable  the  student  to  compre- 
hend the  methods  of  analytical  chemistry  than  to  acquire  particular  proficiency  in 
the  work  of  chemical  analysis.     The  work  is  characterized  by  minuteness  of  explan- 
ation, a  feature  which  will  be  appreciated  by  the  beginner.— Pharmaceutical  t,ra. 

NETTO.  —  The  Theory  of  Substitutions  and  its  Application  to  Algebra. 
By  Dr.  Eugene  Netto,  Professor  of  Mathematics  in  the  University  of 
Giessen.  Revised  by  the  author  and  translated  with  his  permission, 
by  F.  N.  Cole,  Ph.D.,  formerly  Assistant  Professor  of  Mathematics 
in  the  University  of  Michigan,  Professor  of  Mathematics,  Columbia 
University.      8  vo.      301   pages.     Cloth.     $3.00. 

NOW. — Laboratory  Work  in  Physiological  Chemistry.      By  Frederick  G. 

Novy,  Sc.D.,  M.D.,  Junior  Professor  of  Hygiene  and  Physiological 

Chemistry,   University  of    Michigan.      Second    edition,    revised    and 

enlarged.     With  frontispiece  and   24  illustrations.      Octavo.     Cloth, 

$2.00. 

This  book  is  designed  for  directing  laboratory  work  of  medical  students,  and  in 

showing  them  how  to  study  the  physics  and  physiology  of  the  digestive  functions  of 

the  blood,   the  urine  and   other  substances   wiiich  the   body  contains  normally,  or 

which  it   speedily  eliminates  as   effete  material.     The  second  edition  has  appeared 

within  a  very  short   time  after  the  publication  of  the  first.     The  first  chapters  deal 

with  the  facts,  the  carbohydrates  and  proteids.    Then  follow  others  upon  the  saliva, 

the  gastric  juice,  the  pancreatic  secretion,  the  bile,  blood,  milk,  and  urine,  while  the 

closing  chapter  deals  with  a  list  of  reagents. 

While  the  book  is  manifestly  desigired  for  the  use  of  Dr.  Novy's  own  students,  we 
doubt  not  that  other  teachers  will  find  it  a  valuable  aid  in  their  work.  At  the  close 
of  the  volume  are  a  number  of  illustrations  of  the  various  sedimentary  substances 
found  in  the  urine,  taken  from  the  work  of  von  Jaksch. — The  Therapeutic  Gazette 

This  book,  although  now  in  its  second  edition,  is  practically  unknown  to  British 
readers.  Up  to  the  present,  anyone  wishing  to  find  out  how  a  particular  analytical 
method  in  physiological  chemistry  ought  to  be  carried  out,  had  of  necessity  to  refer 
to  a  German  text-book.  This  comparatively  small  book — for  it  only  covers  some 
three  hundred  pages — gives  as  good  a  general  account  of  ordinary  laboratory  methods 
as  any  teacher  or  Student  could  desire.  Although  the  author  refers  in  his  preface  to 
help  derived  from  the  works  of  Salkowski,  Hammaisten  and  others,  it  is  but  fair  to 
say  that  the  book  has  undoubtedly  been  written  by  one  who  has  worked  out  the 
methods  and  knows  the  importance  of  exact  practical  details  —  Edinburgh  Med. 
Jim  1 .,  Si  otland, 


Physiological  chemistry  is  one  of  the  most  important  studies  of  the  medical  curri- 
culum. The  cultivation  of  tnis  field  has  until  recently  been  possible  to  but  few. 
The  rapid  development  of  this  department  of  science  within  a  few  years  past  has 
throv/n  much  and  needed  light  upon  physiological  processes.  It  is  from  this  quarter 
and  from  bacteriological  investigations  that  progress  must  chiefly  be  expected.  The 
rapid  growth  of  this  branch  of  chemistry  is  attended  by  another  result.  It  necessi- 
tates the  frequent  revision  of  text-books.  The  present  edition  of  Dr.  Novy's  valu- 
able book  is  almost  wholly  rewritten.  It  is  representative  of  the  present  state  of 
knowledge  and  is  replete  with  information  of  value  alike  to  student  and  practitioner. 
Few  are  better  prepared  to  write  such  a  book  than  Dr.  Novy,  who  has  himself  done 
much  original  work  in  this  field. — The  Medical  Bulletin,  Philadelphia. 

This  is  a  greatly  enlarged  edition  of  Dr.  Novy's  work  on  Physiological  Chemistry, 
and  contains  a  large  amount  of  new  material  not  found  in  the  former  edition.  It  is 
designed  as  a  text-book  and  guide  for  students  in  experimental  work  in  the  labora- 
tory, and  does  not  therefore  cover  the  same  ground  as  the  works  of  Gamgee,  Lea, 
and  other  authors  of  books  on  physiological  chemistry.  As  a  laboratory  guide  it 
should  be  adopted  by  our  medical  colleges  throughout  the  country,  because  it  is  an 
American  production,  contains  only  such  directions  and  descriptions  as  have  been 
verified  by  actual  practice  with  students,  and  because  it  is  clear,  concise  and  definite 
in  all  its  statements.  Its  nrst  ten  chapters  treat  of  fats,  carbohydrates,  proteins, 
saliva,  gastric  juice,  pancreatic  secretion,  bile,  blood,  milk,  and  urine.  Chapter  xi. 
is  devoted  to  the  quantitative  analysis  of  urine,  milk,  gastric  juice,  and  blood,  while 
chapter  xii.  gives  tables  for  examination  of  urine  and  a  list  of  reagents. — A.m. 
Medico-Surgical  Bulletin,  N.  Y. 

NOVY. — Laboratory  Work  in  Bacteriology.  By  Frederick  G.  Novy,  Sc. 
I).,  M.D.,  Junior  Professor  of  Hygiene  and  Physiological  Chemistry, 
University  of  Michigan.  Second  edition,  entirely  rewritten  and 
enlarged,  563  pages.      Octavo.      $3.00. 

As  a  teacher  of  bacteriology,  the  author  has  had  extensive  experience,  and  the 
second  edition  of  his  book  will  be  highly  prized  by  students  for  its  practical  service 
and  thoroughness.  The  methods  of  investigation  described  are  mainly  those  which 
have  been  employed  in  the  hygienic  laboratory  or  the  University  of  Michigan,  and 
they  have  stood  the  test  of  practical  demonstration  and  usefulness.  One  of  the 
moit  interesting  parts  of  the  book  is  the  chapter  on  the  chemistry  of  bacteria,  and 
the  general  reader  cannot  fail  to  obtain  (rom  it  a  clear  understanding  of  the  com- 
plex changes  induced  by  these  minute  organisms.  The  functions  of  the  various 
ferments  are  also  very  cleverly  discussed.  An  enumeration  of  the  chapter  headings 
will  serve  to  show  the  scope  of  the  work  :  Form  and  Classification  of  Bacteria ;  Size 
and  Structure  of  Bacterial  Cell ;  Life  History  of  Bacteria  ;  Environment  of  Bacteria ; 
Chemistry  of  Bacteria;  the  Microscope;  Cultivation  of  Bacteria;  Non-Pathogenic 
Bacteria;  Bouillon,  Agar,  Milk  and  Modified  Media,  the  Incubator  and  Accessories; 
Relation  of  Bacteria  to  Disease — Methods  of  Infection  and  Examination;  Patho- 
genic Bacteria;  Yeasts,  Moulds  and  Streptotrices ;  Examination  of  Water,  Soil  and 
Air;  Special  Methods  of  Work.  To  the  latter  subject,  two  chapters  are  devoted, 
in  which  are  very  fully  outlined  various  special  methods  of  value  to  advanced 
students.— Pharmaceutical  Era,  N.  Y. 

STRUMPELL. — Short  Guide  for  the  Clinical  Examination  of  Patients. 
Compiled  for  the  Practical  Students  of  the  Clinic,  by  Professor  Dr. 
Adolf  Striimpell,  Director  of  the  Medical  Clinic  in  Erlangen.  Trans- 
lated by  permission  from  the  third  German  edition,  by  Jos.  L.  Abt. 
Cloth,  39  pages,  35  cents. 

Preface  to  the  Second  Edition. — The  second  edition  of  this  book  has  been 
improved  by  me  in  several  parts,  and  particularly  the  sections  treating  of  the  exam- 
ination of  the  stomach  and  nervous  system  nave  been  slightly  extended.  The  author 
trusts  that  the  book  may  also  fulfill  its  purpose  in  the  future  in  assisting  the  student 
to  learn  a  systematic  examination  of  the  patient,  and  to  impress  on  him  the  most 
important  requisite  means  and  methods. 

SUNDERLAND.—  One  Upward  Look  Each  Day.  Poems  of  Hope  and 
Faith.  Selected  by  J.  T.  Sunderland.  Third  Edition,  16  mo. 
White  Binding,  30  cents;    Cloth,  40  cents;  Full  morocco,  75   cents. 

SUNDERLAND-t?)w'w  of  Gold.  Some  Thoughts  and  a  Brief  Prayer 
For  Each  Day  of  the  Months.  Designed  as  Daily  Helps  in  the 
Higher  Life.  Compiled  by  J.  T.  Sunderland.  White  Binding,  35 
cents. 


WARTHIN. — Practical  Pathology  for  Students  and  Physicians.  A 
Manual  of  Laboratory  and  Post- Mortem  Techuic,  Designed  Espe- 
cially for  the  Use  of  Junior  and  Senior  Students  in  Pathology  at 
the  University  of  Michigan.  By  Aldred  Scott  Warthin,  Ph.D.,  M. 
D.,  Instructor  in  Pathology,  University  of  Michigan.  Octavo.  234 
pages.     Cloth.  $1.50. 

We  have  carefully  examined  this  book,  and  our  advice  to  every  student  and  prac- 
titioner of  medicine  is — buy  it.  You  will  never  regret  having  invested  your  money  in 
it.  and  you  will  acquire  such  a  large  fund  of  information  that  the  study  of  pathology 
will  become  a  pleasure  instead  of  the  drudgery  which  it  sc  unfortunately  seems  to 
be  in  many  cases. 

Part  I.  of  this  book,  embracing  some  103  pages,  deals  with  the  materials,  which 
includes  the  proper  examination  and  notation  of  the  gross  changes  which  have 
occurred  in  every  part  of  the  body.  In  fact  it  is  a  complete  expose  of  what  a  com 
plete  and  accurate  autopsy  should  be,  the  observance  of  which  is  oftener  followed 
in  the  breach  than  in  the  actuality.  Part  II.,  which  includes  134  pages,  deals  with 
the  treatment  of  the  material.  This  is  a  very  important  part  of  the  work,  as  it  gives 
explicit  directions  in  regard  to  the  instruments  to  use,  stains  and  staining  methods, 
drawing,  the  preservation  of  specimens,  Hardening  methods,  in  fact,  of  all  those 
technical  points  connected  with  practical  pathological  microscopy.  The  examina- 
tion of  fresh  specimens,  injections,  methods  fixing  specimens-  as  well  as  special 
staining  methods  are  taken  up.  In  fact,  space  forbids  us  to  give  the  entire,  which 
are  most  valuable  in  every  detail.— St.  Loui*  Medical  and  Surgical  Journal. 

WATSON. —  Tables  for  the  Calculation  of  Simple  or  Compound  Interest 
and  Discount  and  the  Averaging  of  Accounts.  The  Values  of 
Annuities,  Leases,  Interest  in  Estates  and  the  Accumulations  and 
Values  of  Investments  at  Simple  or  Compound  Interest  for  all  Kates 
and  Periods;  also  Tables  for  the  Conversion  of  Securities  and  Value 
of  Stocks  and  Bonds.  With  full  Explanation  for  Use.  By  James 
C.  Watson,  Ph.D.,  LL.D.     Quarto.      Cloth,  $2.50. 

A  book  most  valuable  to  bankers,  brokers,  trustees,  guardians,  judges,  lawyers, 
accountants,  and  all  concerned  in  the  computation  of  interest,  the  division  and  set- 
tlement of  estates,  the  negotiation  of  securities,  or  the  borrowing  and  lending  of 
money,  is  the  above  work  of  the  late  Professor  James  C.  Watson,  formerly  Director 
of  the  Observatories  and  Professor  of  Astronomy  at  the  Universities  of  Michigan 
and  Wisconsin,  and  Actuary  of  the  Michigan  Mutual  Life  Insurance  Company. 

It  contains,  in  addition  to  the  usual  tables  for  the  calculation  of  simple  or  com- 
pound interest  and  discount,  many  tables  of  remarkable  value,  not  found  elsewhere, 
for  the  averaging  of  accoutns,  the  values  of  annuities,  leases,  interests  in  estates, 
and  the  accumulations  and  values  of  investments;  also  tables  for  the  conversion  of 
securities,  and  the  values  of  stocks  and  bonds. 

There  are  also  given  very  full  and  clear  explanations  of  the  principles  involved  in 
financial  transactions,  and  a  great  variety  of  miscellaneous  examples  are  worked 
out  in  detail  to  illustrate  the  problems  arising  in  interest,  discount,  partial  payments, 
averaging  of  accounts,  present  values,  annuities  of  different  kinds,  annual  payments 
for  a  future  expectation  (as  in  life  insurance),  or  for  a  sinking  fund,  conversion  of 
securities,  values  of  stocks  and  bonds,  and  life  interests. 

This  book  was  issued  from  the  press  under  the  author's  careful  supervision. 
Professor  Watson  was  noted  for  his  clear  insight  into  problems  involving  computa- 
tions, and  also  for  his  wonderful  ability  in  presenting  the  method  of  solution  of  such 
problems  in  a  plain  and  simple  manner.  The  varied  array  of  practical  examples 
given  in  connection  with  his  "Table  "  shows  these  facts  in  a'remarkable  manner. 
This  book  provides,  for  those  least  expert  in  calculations,  the  means  of  avoiding 
mistakes  likely  to  occur ;  and  for  the  man  engrossed  in  the  cares  of  business,  the 
of  making  for  himself,  with  entire  accuracy,  the  calculation  which  he  may 
need,  at  the  moment  when  it  is  needed. 

WRENTMORE-GOULDING.—  A  Text-Book  of  Elementary  Mechan- 
ical Drawing  for  Use  in  Office  or  School.  By  Clarence  G.  Wrent- 
more,  P.S.,  C.E.,  and  Herbert  J.  Gould ing,  B.S.,  M.E.,  Instructors 
in  Descriptive  Geometry  and  Drawing  at  the  University  of  Michigan. 
Quarto.      109  pages  and  165  cuts.      $1.00. 

Thi  book  i  intended  for  a  beginners  course  in  Elementary  Mechanical  Drawing 
for  the  office  ami  school.  Illustrations  have  not  been  spared,  and  the  explanations 
have  been  made  in  a  clear  and  concise  manner  for  the  purpose  of  bringing  the  stu- 


othe  desired  results  by  the  shortest  route  consistent  with  the  imparting  of  an 
accurate  knowledge  of  the  subject. 

The  first  chapter  is  devoted  to  Materials  and  Instruments;  the  second  chapter, 
Mechanical  Construction;  third  chapter,  Penciling.  Inking,  Tinting;  fourth  chap- 
ter, Linear  Perspective;  fifth  chapter,  Teeth  of  Grass. 

WRENTMORE.-Z'/flw  Alphabets  for  Office  and  School.  Selected  by 
C.  G.  Wrentmore,  B.S.,  C.E.,  Instructor  in  Descriptive  Geometry 
and  Drawing,  University  of  Michigan.  Oblong,  19  plates.  Half 
leather,  75  cents. 

Souvenir  of  the  University  of  Michigan,  Ann  Arbor.  Containing  38 
photo-gravures  of  President  James  B.  Angell,  prom.nent  University 
Buildings,  Fraternity  Houses,  Churches,  Views  of  Ann  Arbor,  Etc., 
Etc.     Done  up  in  blue  silk  cloth  binding.     Price,  50  cents,  postpaid. 

Physical  Laboratory  Note  Book. — A  Note  Book  for  the  Physical  Lab- 
oratory. Designed  to  be  used  in  connection  with  Chute's  Physical 
Laboratory  Manual.  Contains  full  directions  for  keeping  a  Physical 
Laboratory  Note  Book.  112  pages  of  excellent  writing  paper,  ruled 
in  cross  sections,  Metric  System,  size  7x9^  inches.  Bound  in  full 
canvass,  leather  corners.  Price,  by  mail,  30  cents.  Special  prices 
to  Schools  furnished  on  application. 

Botanical  Laboratory  Note  Book. — A  Note  Book  for  the  Botajiical  Lab- 
oratory. Contains  directions  for  Botanical  Laboratory.  200  pages 
of  best  writing  paper,  ruled  with  top  margins.  Pocket  on  inside  of 
front  cover  for  drawing  cards.  Bound  in  substantial  cloth  cover  and 
leather  back.  Size  6x  9/^.  Price,  by  mail,  35  cents.  Special  prices 
to  schools  furnished  on  application. 


QP519  N85 

Hovy,  F.G. 
Laboratory  work  in  physiological 


chemistry* 


